Optical cytometry

ABSTRACT

The present invention provides optical systems and methods for determining a characteristic of a cell, such as cell type, cellular response to a biochemical event, biological state and the like. The methods typically involve using interferometry to observe membrane properties in a cell and then use this information to determine one or more characteristics of a cell. The methods of the invention are useful for applications such as drug screening as well as diagnostic techniques.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/088,992, filed Nov. 25, 2013, which is a continuation applicationthat claims the benefit under 35 U.S.C. § 120 of U.S. patent applicationSer. No. 12/436,702, filed May 6, 2009, the contents of all of which areincorporated herein by reference. This application is related to U.S.patent application Ser. No. 11/077,266 filed Mar. 9, 2005, the contentsof which is incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.CA090571, CA107300, and GM074509, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to interferometric systems, materials andtechniques that can used to examine one or more cells.

2. Description of Related Art

Cells are capable of many complex functions such as motility, cell-cellcommunication and the synthesis of a wide variety of biologically activemolecules. Cell membranes play a crucial role in many of these functionsin part due to their ability to adopt a wide variety of morphologicalconfigurations, configurations which depend on factors such as cellularphysiology as well as cell type and lineage specific functions.

Cell membranes and other physical structures of cells are complex anddynamic, with cytoskeletal elements oriented in many directions, andthus their mechanical properties are highly anisotropic, and vary widelyamong individual cells within a population (see, e.g. Smith et al.,(2003) American Journal of Physiology-Lung Cellular and MolecularPhysiology 285, L456-L463; Hu et al., (2003) American Journal ofPhysiology-Cell Physiology 285, C1082-C1090; Fabry et al., (2001)Physical Review Letters 8714; and Fabry et al., (2001) Journal ofApplied Physiology 91, 986-994). The degree and significance of thismechanical anisotropy and its population variances is poorlycharacterized, however, due to methodological limitations of existingnano-mechanical probing techniques.

Existing cytometric approaches, such as those using AFM (see, e.g.Mahaffy et al., (2004) Biophysical Journal 86, 1777-1793), andhigh-magnification particle tracking microrheology (see, e.g. Weihs etal., (2006) Biophysical Journal 91, 4296-4305), are simply too slow toadequately measure the number of individual cells required forpopulation comparisons. On the other hand, wide-field magnetic/opticalbead tracking methods, which rely on beads fixed to the cell surface,can only track the probe with sufficient accuracy (tens of nanometers(see, e.g. Mijailovich et al., (2002) Journal of Applied Physiology 93,1429-1436; and Cheezum et al., (2001) Biophysical Journal 81, 2378-2388)in two dimensions (the x-y plane perpendicular to the objective).

In view of the limitations with existing cytometric technologies, thereis a specific need to extend probe-based mechanical measurements intoall three dimensions, while retaining measurement accuracy and highthroughput. In addition, there is a general need in the art foroptimized methods of observing and/or determining one or morecharacteristics of a cell (e.g., determining the physiological status orbiological state of a cell; determining the cell type of a cell;determining the response of a cell to a biochemical event; etc.). Theinstant invention addresses these needs.

SUMMARY OF THE INVENTION

Embodiments of the invention include, for example, systems, methods andmaterials that can be used to determine one or more characteristics of adeformable material such as the membrane of a cell. Illustrativeembodiments of the invention involve observing one or more properties ofa cell with an interferometer and then using these observations tocharacterize one or more aspects of cellular physiology. Such propertiesinclude for example: observations of cell and/or cell membrane motion byobserving membranes coated with micromirrors; observations cell and/orcell membrane motion in the absence of micromirrors via real-time phasemeasurements; as well as observations of optical cell thickness (celldensity), cell volume, and the like. The systems and methods of theinvention can be used for example to obtain information useful for awide variety of biomedical applications such as diagnostic procedures(e.g. to identify a pathological condition in an individual from which acell is obtained) as well as drug screening assays (e.g. to test andidentify agents capable of modulating a cell's physiology). The systemsand methods of the invention can also be used, for example, to measurecell responses to any physical change in the local environment, such aschange in temperature, pH, force application, and cell density andneighboring cell effects, such as touching, cytokine related signaling,vibration sensing, and others.

The invention disclosed herein has a number of embodiments. Embodimentsof the invention include, for example, systems and/or methods forobserving a property of a deformable material comprising: a microscopecapable of measuring a feature of interest in a sample; a detectoroperatively coupled to the microscope; a sample assembly comprising anobservation chamber adapted to contain the deformable material; and aplurality of reflective microparticles capable of adhering to thedeformable material, wherein the average diameter of the reflectivemicroparticles is between 0.5 μm and 30 μm. In certain embodiments, themicroscope is a confocal microscope. In other embodiments, themicroscope is an interference microscope capable of observinginterference fringes through a fluid medium. The systems and/or methodsof the invention can be used to obtain a variety of types ofinformation, for example information relating to an axial position of amagnetic reflective microparticle disposed on or proximal to adeformable material; and/or information relating to a z motion of amagnetic reflective microparticle disposed on or proximal to thedeformable material. Certain embodiments of systems and/or methodsdisclosed herein comprise optical profiling techniques such as confocalor digital holography, spectrally resolved interferometry, wavelengthscanning interferometry, digital holography and the like.

A general embodiment of the invention is a system for observing aproperty of a deformable material comprising: a microscope having aMichelson interference objective; a detector such as a camera (e.g. astill camera, a video camera, charge coupled devices (CCD) and the like)operatively coupled to the microscope; a sample assembly comprising anobservation chamber adapted to contain the deformable material; areference assembly comprising a reference chamber; a plurality ofreflective magnetic microparticles capable of adhering to the deformablematerial; and a magnet disposed below the observation chamber andoriented coaxially with an optical axis; wherein the magnet isoperatively coupled to a motorized micrometer and adapted to exert amagnetic force on a magnetic reflective microparticle adhered to thesurface of the deformable material. Such general embodiments arenon-limiting as the systems disclosed herein can adopt a variety ofconfigurations. Embodiments of the invention further include methods forobserving a property of a deformable material using the systemsdisclosed herein. While cellular membranes are the focus of thefollowing disclosure relating to these systems and methods, those ofskill in the art understand that a wide variety of other deformablematerials can be observed and/or characterized using embodiments of theinvention disclosed herein.

One typical embodiment of the invention is a system for obtaining animage of a cell (and/or cells within a population simultaneously)comprising: a microscope having a Michelson interference objective; acamera operatively coupled to the microscope; a sample assemblycomprising an observation chamber adapted to contain the cell; areference assembly comprising a reference chamber adapted to contain afluid; and a plurality of reflective microparticles capable of adheringto the cell, wherein the average diameter of the reflectivemicroparticles is between 0.5 μm and 30 μm (e.g. spherical magneticmicroparticles having an average diameter of between 1 μm and 15 μm or 5μm and 10 μm etc.). Optionally the reflective microparticles comprise agradient index (GRIN) spherical lens. In certain embodiment of theinvention, the reference assembly further comprises: a first opticalwindow; a first housing element adapted to hold the first opticalwindow; a second optical window; a second housing element adapted tohold the second optical window; and a plurality of spherical spacerelements disposable between the first optical window and the secondoptical window and adapted to separate the first and second opticalwindows to a defined distance. In embodiments of the invention, thesample assembly can further comprise: a viewing window and a firsthousing element adapted to hold the viewing window, wherein thethickness of the viewing window is equivalent to the combined thicknessof the first and second optical windows in the reference assembly.Moreover, in such embodiments of the invention the sample assembly canalso comprise a plurality of spherical spacer elements disposablebetween the viewing window and a top portion of the observation chamberand adapted to separate the viewing window and the top portion of theobservation chamber to a defined distance that is equivalent to thedefined distance between the first and second optical windows in thereference assembly. In typical embodiments of the invention, a surfaceof the observation chamber (e.g. the surface that is farthest away fromthe detection camera lens) is reflective.

Embodiments of the invention include a variety of permutations of thesesystems. For example, in certain embodiments, the observation chambercomprises at least one perfusion conduit adapted to circulate a cellmedia within the chamber. Optionally the observation chamber isoperatively coupled to other elements adapted to control the environmentin which the cell is disposed such as heating elements and the like.Some embodiments of the invention also include a magnet disposed belowthe observation chamber and oriented coaxially with an optical axis.Typically in such embodiments, the magnet is operatively coupled to amotorized micrometer and adapted to exert a magnetic force of between 0Newtons and 5 nanoNewtons on a magnetic reflective microparticle adheredto the surface of a cell. In some embodiments of the invention, themagnet is typically adapted to generate a magnetic field of between 200Gauss and 3 kiloGauss. In embodiments of the invention, the magnet istypically adapted to generate a magnetic field gradient range of between300,000 to 800,000 Gauss/meter. Typical embodiments of the inventionfurther comprise a processor element and a memory storage elementadapted to process and store one or more images of the cell.

Related embodiments of the invention include methods of using thesystems disclosed herein. One such embodiment of the invention is amethod for observing a property of a cell, the method comprising:adhering a reflective magnetic microparticle to the cell; placing thecell in a cell observation chamber of an optical microscope having aMichelson interference objective; exposing the cell coated with themicroparticle to a magnetic field; and then using the Michelsoninterference objective to observe the movement of the microparticleadhered to the cell in response to the applied magnetic field, whereinthe movement of the reflective microparticle adhered to the cellcorrelates to a property of the cell, so that a property of the cell isobserved.

A variety of methodological embodiments are contemplated. For example,certain methodological embodiments of the invention are performed usinga system comprising: a camera operatively coupled to the microscope; asample assembly comprising an observation chamber adapted to contain thecell; a reference assembly comprising a reference chamber adapted tocontain a fluid; a memory storage element adapted to store one or moreimages of the cell; and a processor element adapted to process one ormore images of the cell.

The methods of the invention can be used to obtain a wide variety ofinformation relating to one or more cellular properties. For example, incertain embodiments of the invention, the method can be used to observean optical thickness of a live cell in an aqueous medium. Alternatively,the method can be used to observe a cell mass property of a live cell inan aqueous medium. In some embodiments of the invention, the method isused to observe a viscoelastic property of a live cell in an aqueousmedium. Optionally, the method is used to observe a population of livecells, for example to observe resting and dynamic responses to stimuliin a population of live cells. Typically in these methods, the propertyis observed in response to the cell's exposure to a stimulus such as themagnetic field applied to the cell and/or a composition introduced intothe cell's media. Optionally the methods further comprise removing thecell from the observation chamber and manipulating the cell for afurther analysis. In certain embodiments of the invention, the method isused to obtain information comprising a cell specific profile of a livecell in an aqueous medium and to then store this information in thememory storage element.

Embodiments of the invention also include a reflective microparticlecomprising a gradient index (GRIN) spherical lens. Those of skill in theart will further understand that other types of particles can be used inembodiments of the invention. For example, relatively uniform and flatcylindrical 8-10 micron nickel beads (e.g. ones made byphotoresist/electroplating methods) can be used in various embodimentsof the invention. Optionally such microparticles are coupled to aflexible tether and/or an optical fiber and/or is operatively coupled toan endoscope. In certain embodiments of the invention, thismicroparticle comprises a plurality of material layers, whereinrefractive indices of the material layers decrease from the center ofthe microparticle.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of an embodiment of an interferometricmicroscope. This microscope embodiment can typically accommodate a 5×and 20× long-working distance microscope objective. Embodiments of theinvention can use other objective lenses known in the art, for examplethose used for higher and lower magnification (e.g. 50× etc.). Below theobjective is the Michelson interferometer with an adjustable mirror inthe reference arm. A fluid compensation cell is positioned in theinterferometer's reference arm to permit measurements inside themedia-filled cell chamber. In embodiments of the invention, thedimensions of the compensation cell can be adjusted to exactly match theoptical path length between the test and reference arms. With thissystem, cells can be evaluated in a sealed environmental chambermaintained at 5% CO₂, 37° C., with infusion ports for exchanging mediaand the introduction of drugs and other chemicals. Typically thisembodiment, a cylindrical rare-earth magnet mounted on a micrometer istypically positioned below the cell chamber. The magnitude of themagnetic force applied to the nickel microreflectors inside the cellchamber is then adjusted by varying the distance between the magnet poleface and the sample. FIG. 1B shows a side view schematic ofinterferometric microscope system elements that can be used withembodiments of the invention. FIG. 1C shows photographs of non-magneticstainless steel reference and sample assembly elements that can be usedwith embodiments of the invention. FIG. 1D shows a schematic of sampleassembly elements (e.g. the arrangement of windows in the assembly thatcan be used with embodiments of the invention. FIG. 1E shows a schematicof reference assembly elements that can be used with embodiments of theinvention. FIG. 1F shows photographs of non-magnetic steel sampleassembly elements that can be used with embodiments of the invention.FIG. 1G shows a schematic of sample assembly elements that can be usedwith embodiments of the invention.

FIG. 2A shows a schematic of the geometry of the force-indentation testsusing a 40 μm thick polyacrylamide (PA) gel to simulate the cell body.FIG. 2B provides a graph of a showing the deflection of a 7 μm nickelmicroreflector into a 0.05% crosslinker PA gel, under a series ofdecreasing forces; A-6.6, 5.3 and 4.2 nN; B—4.2, 3.3, 2.8 nN; C— 2.4,1.9 and 1.6 nN. Force-distance measurement can be fitted to the Hertzcontact model for a spherical indenter, from which the gel's elasticmodulus is calculated. FIG. 2C provides individual measurements on a0.05% and a 0.15% crosslinker gel show the correspondence between theforce-deflection behavior of the microreflectors and that predicted bythe Hertz model. FIG. 2D provides a graph showing that the measuredvalues for Young's modulus were linearly proportional to the crosslinkerconcentration, as expected, and the range of absolute values(1,530+/−600 Pa and 4,020+/−1,300 Pa) agree well with similarmeasurements by others using AFM and bulk techniques (see, e.g. Mahaffyet al., (2004) Biophysical Journal 86, 1777-1793; Mahaffy et al., (2000)Physical Review Letters 85, 880-883; Engler et al., (2004) BiophysicalJournal 86, 617-628).

FIG. 3A shows the intensity image of an NIH 3T3 fibroblast (Top left)with a microreflector positioned on the cell membrane (arrow). Thecorresponding vertical scanning interferometry (VSI) height profile(middle) includes only the microreflector since the cell body isminimally reflective and can be seen and/or calculated out inembodiments of the invention. The phase-shifting (PSI) interferometricimage (right), shows the cell body, where apparent height corresponds toincreased optical path length due to the higher index of refraction ofthe cytoplasm versus the surrounding media. The use of PSI measurementswith this apparatus is detailed in (see, e.g. Reed et al., (2006) Proc.SPIE Int. Soc. Optical Eng., 6293: 629301). The microreflector is opaqueand does not appear in the PSI image because it is not the focus in theoptical field being examined. Below: The VSI and PSI height profiles arerendered in 3D for clarity. FIG. 3B shows an intensity image of NIH 3T3fibroblasts coated with nickel micromirror beads in the cell chamber,taken at 10× magnification. The field of view is 600×460 μm. The VSIinterferometric image is overlaid in blue, showing the detection of 103microreflectors (example indicated with white circle).

FIG. 4 shows a force-distance curve showing the deflection of a 10 μmnickel microreflector into a single HEK293T cell. The viscoelasticnature of the cell body is apparent from the delay between the onset offorce change and the time required to reach an equilibrium deflection(creep). This creep phenomenon is not captured by the time-invariantHertz contact model. A simple three-factor viscoelastic solid model,represented by the mechanical spring and dashpot model (inset),describes the observed behavior more completely. This model contains aninstantaneous elastic constant, E₁, and a time-delayed elastic constant,E₂. The time delay is governed by the magnitude of E₂ and the viscosity,η. The three viscoelastic constants can be calculated by fitting theobserved force-deflection curve to a version of the three-factor modelapplicable to spherical indenter geometry (see, e.g. Cheng et al.,(2005) Mechanics of Materials 37, 213-226). Curves are fitted using theLevenberg-Marquardt non-linear least squares procedure.

FIG. 5 shows the population distribution of the three viscoelasticconstants determined for populations of NIH 3T3 and HEK 293Tfibroblasts. The error bars indicate standard error of the mean, and *indicates statistical significance at the >95% level. The mean of thelog-transformed distribution of E₁ was 3.45 for NIH3T3 and 3.33 forHEK293T fibroblasts (p=0.10). The means of the log-transformeddistributions of E₂ were 3.06 and 2.90 (p=0.03), and the means of thelog-transformed viscous constants η were 4.11 and 4.00 (p=0.17),respectively.

FIG. 6A shows the intensity image (left) of NIH3T3 cells withmicroreflectors in place, before and after treatment with 1 μmcytochalasin B. FIG. 6B shows the force-displacement curves of fourindividual microreflectors before and after treatment shows the changein viscoelastic behavior in response to normal force applied for 100seconds (t=0-100 on the graphs). Probes 2 and 3 show a clear decrease instiffness, while probe 4 shows a change in elastic rebound behavior, andprobe 1 appears to be unchanged. The force generated on the cell by eachmicroreflector is a function of the probe's total mass: (1) radius+3.85μm, force=180 pN; (2) radius=3.90 μm, force=190 pN; (3) radius=3.40 μm,force=130 pN; (4) radius=4.75 μm, force=340 pN.

FIG. 7 Upper left panels show LCI interferometric images of a live NIH3T3 fibroblast taken two seconds apart, before and after the applicationof force by two magnetic microspheres on their surface (indicated byblack disks). The optical thickness cross-sections are displayed to theright. The change in optical thickness between the two images is readilyapparent in the differential LCI image, below, created by subtractingthe bottom from the top LCI image. As shown by the associated panels onthe right, the optical thickness of the cell body ranges from 0-400 nmand the change in optical thickness detectable in the differential LCIimage ranges from −6 to +8 nm.

FIGS. 8A-8C show intensity (left) and LCI interferometric (right) imagesof a single NIH3T3 cell with two magnetic microspheres on the cellsurface. As force was applied to the probes, the change in optical pathlength in the regions directly surrounding each probe (A1 and B1), andthe adjacent regions (A2 and B2) was tracked. A 200 pN peak-to-trough,0.05 Hz cyclical force was applied to the microspheres for 200 s. FIG.8D shows a schematic of the geometry used for calibrating the magneticforce applied to each microreflector. FIG. 8E shows a force calibrationcurve and associated magnetic field, as a function of distance betweenthe magnet face and the sample. FIG. 8F shows the average time-varyingoptical thickness measurements for four regions within the cell depictedin FIG. 8A. Individual data points are collected at two secondintervals, for a 0.5 Hz sampling frequency. For clarity, the data havebeen band pass filtered with a 0.05 Hz center frequency.

FIG. 9 shows differential LCI images of three indentation cycles at 20,120 and 200 s. The top panel of images shows the effect of probeindentation immediately after force is applied, and the bottom panelshows the corresponding rebound after force is removed. The pattern andmagnitude of material redistribution appears to change with time.

FIGS. 10A and 10 b show the effect of 0.05 Hz cyclical loading on theadjacent cellular regions is seen as a change in average opticalthickness. The cyclic amplitude of the time-varying change opticalthickness of regions A1 and A2 (FIG. 10A) evaluated at frequency=0.05 Hzshow the relationship between the driven and undriven portions of thecell body (top). Similar behavior is seen in regions B1 and B2 (FIG.10B). The regions adjacent to the probes show a clear response,indicating that the strain field within the cell body extends severalprobe diameters laterally. Individual data points are collected at twosecond intervals, for a 0.5 Hz sampling frequency. For clarity, the datahave been band pass filtered with a 0.05 Hz center frequency.

FIG. 11 shows the Differential LCI comparison of material distributionsafter t=200 s of cyclically-applied force Δ200/0 (left panel) and at 200s after cessation of force t=400 s; Δ400/200 (right panel). Thepositions of the microspheres are indicated by black circles. Arrowsdenote regions of material redistribution within the cell.

FIG. 12A shows a graph of a range of a typical range of forces appliedto a 5-10 micron reflector on a cell over time and the associateddeflection. FIG. 12B shows a graph of forces applied to a cell over timeand the associated active responses from the cell.

FIGS. 13A and 13B show photographs of cellular environments that can beused with embodiments of the invention and can be made for example byphotoresist deposition processes known in the art. Such environments(with “holes” (e.g. nanowells or microwells) of an appropriate size) cancreate structures that facilitate uniform cell packing analysis. Inthese FIG. 13B, cells labeled 1, 2, 3 are in 10 micron-sized wells.

FIG. 14A shows a schematic of one illustrative way to fabricatemicromirrors/reflectors from curable polymers. FIG. 14B shows aphotograph of reflectors fabricated in this manner. FIGS. 14C-14E showschematics of other processes known in the art (e.g. sputtering, spincoating, photoresist and electroplating technologies etc.) that can beused to generate micromirrors/reflector embodiments of the invention.

FIG. 15A shows a schematic of a Luneberg lens with a radially varyingindex of refraction (N), such that an entering signal will be refractedinto a nearly elliptical path to a point on the opposite surface of thesphere. FIG. 15B shows a schematic of a 6-shell sphere, with refractiveindex decreasing with each layer from center, one which approximates theideal Luneberg lens.

FIG. 16 shows one embodiment of a illustrative magnetically drivenoptical bead probe and elements typically associated with such probes.

FIG. 17 shows one embodiment of a illustrative computer system that canbe used with embodiments of the invention.

DETAILED DESCRIPTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings may be defined herein for clarity and/or for ready reference,and the inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. A number of termsare defined below.

As used herein, the term “membrane” refers to a barrier between thecytoplasm of a cell and the extracellular environment, or between theinterior of a subcellular organelle and the cytoplasm of a cell. A“membrane” includes a eukaryotic animal, fungal, or yeast cell membraneor cell wall, which generally comprises a lipid bilayer and may includeother components such as polypeptides, glycoproteins, lipoproteins, andpolysaccharides; a plant cell wall, which generally comprises cellulose,and other components such as lignin, pectins, and hemi-celluloses; abacterial cell wall (including cell walls of archaebacteria andeubacteria); and the like. Membranes include artificial as well asnaturally-occurring membranes, such as external cell membranes, nuclearmembranes, mitochondrial membranes, and the membranes of otherorganelles. In embodiments of the invention, many structures can beimaged in isolation and/or within a live cell by changing plane of focus(e.g. because membranes are not completely opaque compared withsurrounding liquid environments).

The term, “cell characteristic” is used according to its art acceptedmeaning and includes for example the biological state of a cell and/orthe cell type of a cell and/or a cell's response to a biochemical event.Typically such characteristics can be correlated with one or morephysiological phenomena such as oncogenic transformation. Membranemovement of the cell is one cellular characteristic that can be observedand then correlated with physiological phenomena. “Biological state” (or“physiological status”) includes, but is not limited to, the status of acell in response to one or more stimuli, controlled cell division (e.g.,mitosis); uncontrolled cell division (e.g., cancerous state); activeprotein synthesis; quiescence; apoptosis; adhesion to a surface;metastasis; and the like.

“Cell type” refers to the role that a cell plays under normalphysiological conditions. Non-limiting examples of cells are cells ofmulticellular organisms, e.g., cells of invertebrates and vertebrates,such as myoblasts, neutrophils, erythrocytes, osteoblasts, chondrocytes,basophils, eosinophils, adipocytes, invertebrate neurons (e.g., Helixaspera), vertebrate neurons, mammalian neurons, adrenomedullary cells,melanocytes, epithelial cells, and endothelial cells; tumor cells of alltypes (e.g., melanoma, myeloid leukemia, carcinomas of the lung, breast,ovaries, colon, kidney, prostate, pancreas and testes); cardiomyocytes,endothelial cells, lymphocytes (e.g. T-cells and B cells), mast cells,vascular intimal cells, hepatocytes, leukocytes including mononuclearleukocytes; stem cells such as hematopoietic stem cells, neural, skin,lung, kidney, liver and myocyte stem cells; osteoclasts, connectivetissue cells, keratinocytes, melanocytes, hepatocytes, and kidney cells.Suitable cells also include known cell lines, including, but not limitedto, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. Cell lines includethose found in ATCC Cell Lines and Hybridomas (8th ed, 1994, or latestedition, or on the world wide web at www.atcc.org), Bacteria andBacteriophages (19th ed., 1996), Yeast (1995), Mycology and Botany (19thed., 1996), and Protists: Algae and Protozoa (18th ed., 1993), availablefrom American Type Culture Co. (Manassas, Va.). In certain embodiments,a specific lineage of cells noted above such as muscle cells arespecifically excluded from an analysis, e.g., the cell is not a musclecell. In certain embodiments, transformed eukaryotic cell lines, such asHEK293 cells, are specifically excluded from an analysis.

The term “biological sample” is used according to its art acceptedmeaning and encompasses for example biological materials (typicallycontaining cells) that are examined in a wide variety of diagnosticand/or monitoring assays known in the art. The definition encompasses invitro samples such as cell cultures and related samples as well as invivo samples such as those obtained from blood and other liquid samplesof biological origin, solid tissue samples such as biopsy specimens orcells derived therefrom and the progeny thereof. The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as polynucleotides orpolypeptides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit relatively autonomous growth, sothat they exhibit an aberrant growth phenotype characterized by asignificant loss of control of cell proliferation and/or extendedsurvival. Cancerous cells are malignant, whereas a tumor or neoplasm canbe benign or malignant.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims. It must also be noted that as used herein and in the appendedclaims, the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a micromirror” includes a plurality of suchmicromirrors and equivalents thereof known to those skilled in the art,and so forth. All numbers recited in the specification and associatedclaims that refer to values that can be numerically characterized with avalue other than a whole number (e.g. the concentration of a compound ina solution) are understood to be modified by the term “about”. Where arange of values is provided, it is understood that each interveningvalue, to the tenth of the unit of the lower limit unless the contextclearly dictates otherwise, between the upper and lower limit of thatrange and any other stated or intervening value in that stated range, isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further the actualpublication dates may be different from those shown and requireindependent verification.

The invention disclosed herein has a number of embodiments. Embodimentsof the invention provide methods, materials and systems for observingand/or characterizing one or more properties of a deformable compositionsuch as the membrane of a cell. Illustrative embodiments of theinvention comprise using interferometry to detect a movement of amembrane of the cell in order to obtain information on and/orcharacterize one or more cellular properties. Illustrative cellularproperties that can be observed by embodiments of the invention caninclude for example cytoskeletal remodeling behavior in response to astimulus, for example a stimulus comprising exposure to a drug or otherbiologically active agent as well as a variety of other factors. In someembodiments of the invention, the phenomena that is observed is onecorresponding to, or associated with, a pathological condition such asaberrant cell division, such as occurs in precancerous and cancerouscells. In some embodiments of the invention, the cell membrane in whichmovement is observed is a membrane of a single cell. In otherembodiments of the invention, the membrane properties of a plurality ofcells are observed. In certain embodiments, the membrane is a membraneof a cell in a tissue. In other embodiments, the membrane is a membraneof a cell within a colony of cells (e.g. an in vitro cell culture ofprimary cells taken from a patient or an established cell line). Intypical embodiments of the invention, the cell is a eukaryotic (e.g.mammalian) cell.

Embodiments of the invention can use a variety of optical profilingmethods to observe and/or characterize one or more properties of adeformable composition. Such methods can comprise for example comparinginformation derived from a scanning interferometry signal for a firstsurface location of a test object (e.g. a mammalian cell) to informationcorresponding to multiple models of the test object. The multiple modelscan then be parameterized by a series of characteristics for the testobject. The derivable information compared in such systems can relate toa shape of the scanning interferometry signal for the first surfacelocation of the test object (e.g. the height of a cell or a populationof cells above a matrix). Such optical profiling methods are typicallyinterferometric, however non-interferometric optical profiling methodscan also be used in embodiments of the invention.

In typical interferometric embodiments of the invention, aninterferometer uses, for example, a Mirau, Michelson or Linnikconfiguration. Certain embodiments of the invention can use objectiveswith specific characteristics such as a transmissive media (TTM)interference objective. In addition, methods and elements associatedwith interferometric technologies including spectrally resolvedinterferometry, wavelength scanning interferometry, digital holographyand the like can be used in embodiments of the invention. While manyinterferometric microscopy systems and methods can be adapted for usewith embodiments of the invention, other embodiments of the inventioncan use scanning optical microscopes, confocal microscopes and the like.An illustrative and non-limiting list of publications that describeoptical profiling methods and materials that can be adapted for use withembodiments of the invention are disclosed for example in U.S. PatentApplication Nos. 20050248770; 20050225769; 20050200856; 20050195405;20050122527; 20050088663; 20040252310; 20050117165; 20030234936;20040066520; 20080018966 and 20050167578, the contents of which areincorporated by reference.

Embodiments of the invention use optical profilometry techniques toprovide for example methods of height measurement, shape measurement, aswell as measures of other modulations in the shapes of cell membranesand other deformable materials. Depending on the shape, size andmaterial of a test object such as a cell or a population off cells,these techniques typically use structured light, focusing properties ofoptics, interference of light, etc., to optimize results in aneconomical and practical way. Moire' techniques, ESPI (electronicspeckle-pattern interferometry), laser scanning, photogrammetry, andinterferometry are illustrative techniques developed for conductingthree-dimensional shape measurements. The technique of white-lightvertical scanning interferometry (VSI) is used to provide data shown inFIG. 3A. VSI, also commonly referred to as white-light interferometry orcoherence radar, is used for imaging small objects, typically those withroughness that does not exceed a few micrometers. VSI methodology isbased on detection of the coherence peak created by two interfering,polychromatic wavefronts. It has many advantages such as absolute depthdiscrimination, fast measurement cycle, and high vertical resolution.One advantage of VSI is the ease with which it can be combined withother measurement techniques, such as phase-shifting interferometry(PSI), which are superior in accuracy but may lack the scanning depth ofVSI. PSI is typically used for measurements of smooth surfaces withsmall changes in profile (see K. Creath, “Temporal Phase MeasurementMethods,” Interferogram Analysis, Institute of Physics Publishing Ltd.,Bristol, 1993, pp. 94-140). VSI is generally used to measure smoothand/or rough surfaces with large interpixel height ranges (K. G. Larkin,“Efficient Nonlinear Algorithm for Envelope Detection in White LightInterferometry,” J. Opt. Soc. Am., A/Vol. 13, 832-843 (1996). Thecombination of VSI and PSI has been used, for example, to measure largesteps with PSI precision (C. Ai, U.S. Pat. No. 5,471,303). The PSIOTFtechnique, which is a particular case of VSI and PSI combination,improves measurements of smooth surfaces in the larger height range(see, e.g. Harasaki et al., “Improved Vertical Scanning Interferometry,”Appl. Opt. 39, 2107-2115, 2000). Typical VSI and PSI systems and methodsare disclosed for example in U.S. Pat. Nos. 5,133,601, 5,471,303 andU.S. Pat. No. 6,449,048, and U.S. Patent Application No. 20020196450,the contents of which are incorporated by reference.

Embodiments of the invention include systems and/or methods forobserving a property of a deformable material comprising: a microscopecapable of measuring a feature of interest in a sample; a detectoroperatively coupled to the microscope; a sample assembly comprising anobservation chamber adapted to contain the deformable material; and aplurality of reflective microparticles capable of adhering to thedeformable material, wherein the average diameter of the reflectivemicroparticles is between 0.5 μm and 30 μm. In certain embodiments, themicroscope is a confocal microscope. In other embodiments, themicroscope is an interference microscope capable of observinginterference fringes and the system further comprises a referenceassembly adapted to substantially match an optical path length of thesample assembly. The systems and/or methods of the invention can be usedto obtain a variety of types of information, for example informationrelating to an axial position of a magnetic reflective microparticlecoating or proximal to a deformable material; and/or informationrelating to a z motion of a magnetic reflective microparticle coating orproximal to the deformable material. In addition, certain embodiments ofsystems and/or methods disclosed herein comprise optical profilingtechniques such as confocal or digital holography, spectrally resolvedinterferometry, wavelength scanning interferometry, digital holographyand the like.

One embodiment of the invention is a system for observing a property ofa deformable material comprising: a microscope; a detector such as apoint detector, a line detector, a microbolometer or a camera (e.g. astill camera, a video camera, charge coupled devices (CCD) other imagecapture devices used microscopy and/or the observation of deformablecompositions such as cell membranes) operatively coupled to themicroscope; a sample assembly comprising an observation chamber adaptedto contain the deformable material; a reference assembly comprising areference chamber; a plurality of reflective magnetic microparticlescapable of adhering to the deformable material; and a magnet disposedbelow the observation chamber and oriented coaxially with an opticalaxis; wherein the magnet is operatively coupled to a motorizedmicrometer and adapted to exert a magnetic force on a magneticreflective microparticle adhered to the surface of the deformablematerial. Embodiments of the invention further include methods forobserving a property of a deformable material using the systemsdisclosed herein. While cellular membranes are the focus of thedisclosure provided herein, those of skill in the art understand that awide variety of other deformable materials can be observed and/orcharacterized using embodiments of the invention disclosed herein (e.g.the polymeric acrylamide materials observed in the Examples below). Inaddition, while many embodiments of the invention use cells coated withmicromirrors (e.g. spherical magnetic micromirrors), others do not. Suchembodiments include for example observations of membranes that are notcoated with micromirrors where membrane motion is observed withreal-time phase measurements of factors such as optical cell thickness(cell density), cell volume and the like. These embodiments of theinvention use the system and methodological elements disclosed simply inthe absence of the micromirrors. One such method is a method forobserving a property of a cell (and/or a population of cells), themethod comprising placing the cell in a cell observation chamber of anoptical microscope having a Michelson interference objective; and usingthis Michelson interference objective to observe the movement of thecell. Other embodiments of the invention can use a Mirau and/or Linnikinterferometric objective system. Typically in such embodiments, themovement the cell correlates to a property of the cell such as celldensity and/or cell volume and the like, and in this way the methodsallow a property of the cell to be observed.

Embodiments of the invention include a system for obtaining an image ofa cell comprising: an interference microscope capable of extractinginformation from interferometric fringes; a detector operatively coupledto the interference microscope; a sample assembly comprising anobservation chamber adapted to contain the cell, a reference assemblyadapted to substantially match an optical path length of the sampleassembly, and a plurality of reflective microparticles capable ofadhering to the cell, wherein the average diameter of the reflectivemicroparticles is between 0.5 mm and 30 mm. One typical embodiment ofthe invention is a system for obtaining an image of a cell comprising: amicroscope having a Michelson interference objective; a cameraoperatively coupled to the microscope; a sample assembly comprising anobservation chamber adapted to contain the cell; a reference assemblycomprising a reference chamber adapted to contain a fluid (e.g. themedia disposed in the observation chamber, RPMI, PBS, water or thelike); and a plurality of reflective microparticles capable of adheringto the cell, wherein the average diameter of the reflectivemicroparticles is between 0.5 μm and 30 μm (e.g. spherical magneticmicroparticles having an average diameter of between 1 μm and 15 μm,between 5 μm and 10 μm etc.). Optionally the reflective microparticlescomprise a gradient index (GRIN) spherical lens.

Embodiments of the system are adapted to use a variety elements andmethods known in the art and/or described herein. For example, while hesample and/or reference chambers typically include a fluid, otherembodiments such that do not need a fluid cell, e.g. a transmissivemedia (TTM) objective (e.g. by using a salt) can also be used inembodiments of the invention. Moreover, in certain embodiments of theinvention, the sample chamber hold the cell is closed while in otherembodiments the cell chamber can be open on top (i.e. does not need alid). Embodiments of the invention typically include matching theoptical path difference between the arms of an interferometric system,typically by controlling the sizes and architecture of the elements thatmake up the sample and reference assemblies. For example, in certainembodiments of the invention, the reference assembly further comprises:a first optical window; a first housing element adapted to hold thefirst optical window; a second optical window; a second housing elementadapted to hold the second optical window; and a plurality of sphericalspacer elements disposable between the first optical window and thesecond optical window and adapted to separate the first and secondoptical windows to a defined distance. This is merely an illustrativeand non-limiting example of one way of accomplishing this goal, andthere are a variety of other ways to match the optical path differencebetween the arms etc. (e.g. in an embodiment where just one plate thatmatches the cell chamber, two wedges can shifted with respect to eachother so that the optical path is varied, different types of spacers canbe used instead of spherical spacer elements etc.).

In embodiments of the invention the sample assembly can furthercomprise: a viewing window and a first housing element adapted to holdthe viewing window, wherein the thickness of the viewing window isequivalent to the combined thickness of the first and second opticalwindows in the reference assembly. Moreover, in such embodiments of theinvention the sample assembly can also comprise a plurality of sphericalspacer elements disposable between the viewing window and a top portionof the observation chamber and adapted to separate the viewing windowand the top portion of the observation chamber to a defined distancethat is equivalent to the defined distance between the first and secondoptical windows in the reference assembly (see, e.g. the schematicrepresentations of such assemblies shown in FIGS. 1A-1F). In typicalembodiments of the invention, a surface of the observation chamber isreflective.

Embodiments of the invention include a variety of permutations of thesesystems. For example, in certain embodiments, the observation chambercomprises at least one perfusion conduit adapted to circulate a cellmedia within the chamber. Some embodiments of the invention also includea magnet disposed below the observation chamber and oriented coaxiallywith an optical axis. Typically in such embodiments, the magnet isoperatively coupled to a motorized micrometer and adapted to exert amagnetic force of between 0 Newtons and 5 nanoNewtons (e.g. 0 to 250 pN,250 pN to 1 nanoN, etc.) on a magnetic reflective microparticle adheredto the surface of the cell. In some embodiments of the invention, themagnet can be adapted to generate a magnetic field of between 200 Gaussand 3 kiloGauss. In embodiments of the invention, the magnet can also beadapted to generate a magnetic field gradient range of between 300,000to 800,000 Gauss/meter. Typical embodiments of the invention furthercomprise a processor element and a memory storage element adapted toprocess and store one or more images of the cell. In certain embodimentsof the invention, the cell is labelled with another marker/probe knownin the art such as a fluorescent marker (e.g. green fluorescent protein)and the system includes optical elements adapted to image these labelledcells. Some embodiments of the invention include additional elementsused to observe cellular properties such as devices and processes (e.g.software based processes) used in FT infrared spectroscopy, Ramanspectroscopy and the like.

Related embodiments of the invention include methods of using thesystems disclosed herein. One such embodiment of the invention is methodfor observing a property of a cell, the method comprising: adhering areflective magnetic microparticle to the cell; placing the cell in acell observation chamber of an optical microscope having a Michelsoninterference objective; exposing the cell coated with the microparticleto a magnetic field; and then using the Michelson interference objectiveto observe the movement of the microparticle adhered to the cell inresponse to the applied magnetic field, wherein the movement of thereflective microparticle adhered to the cell correlates to a property ofthe cell, so that a property of the cell is observed.

A variety of methodological embodiments are contemplated. For example,certain methodological embodiments of the invention are performed usinga system comprising: a camera operatively coupled to the microscope; asample assembly comprising an observation chamber adapted to contain thecell; a reference assembly comprising a reference chamber adapted tocontain a fluid; a memory storage element adapted to store one or moreimages of the cell; and a processor element adapted to process one ormore images of the cell.

The methods of the invention can be used to obtain a wide variety ofinformation relating to one or more cellular properties. For example, incertain embodiments of the invention, the method can be used for exampleto observe an optical thickness of a live cell in an aqueous medium.Embodiments of the invention can be used to measure the opticalthickness of a live cell in liquid (i.e. culture medium) to 1 nmvertical resolution with an image capture rate of 1 every 11 secs (canbe increased to 1 in 1/1000th of a second with modifications) for allcells in the field of view. This observation provides useful informationand comprises, for example, a measure of the proteins, nucleic acids andother molecules in the cell that retard the return of the interferometerlight back to the CCD detector camera on a pixel-by-pixel basis acrossthe horizontal axis of a cell body within the field of view.

Alternatively, the method can be used to observe a cell mass property ofa live cell in an aqueous medium. For example, cell mass in liquid canbe calculated for each cell from observations obtained from embodimentsof the systems disclosed herein. By collecting such calculations over aperiod of time, adaptive and/or maladaptive changes in cell opticalthickness (mass) can be evaluated in response to environmental (i.e.drugs, cytokines) or cell internal (i.e. genetic manipulations by RNAi,gene knockout, over-expression technologies) alterations. For example,one can use measurements of the motion(s) of one or more opticallyreflective object(s) (i.e. beads, mirrors) on the surface of a cell andfor all cells within the field of view in a resting state, to observeresponse to drugs, genetic alterations, and/or in response to magneticforce application. From this information, one can then, for example,derive biophysical parameters for each cell in the field, such asviscoelasticity (typically using certain calculations known in the artand/or disclosed in the Examples below). In this way, artisans canobserve cell properties under changing conditions over time.

In yet another embodiment of the invention cell motion “signatures” canbe derived for each individual cell in a population at rest or inresponse to a perturbation. Motion can be parameterized across a cellbody, or almost instantaneous measurements of strain across a cell canbe made. Transient versus permanent alterations in cell appearance andoptical thickness can be determined from perturbations. Cell exhaustion(no more responses) and death (by incorporatingimmunofluorescence-detecting objectives and vital stains such asAnnexinV and/or propidium iodide and others) can be evaluated by repeator extreme stimulations. The cell cycle can (in concept) be visualizedand biophysical properties evaluated (i.e. by force required to indentthe cell membrane with a reflector during cell division).

In yet another embodiment of the invention, individual live cells withunique properties can be isolated and recovered from the field of viewbecause their position(s) are identified in the interferometer field ofview. Further manipulations such as recovering an observed cell foradditional analyses are contemplated. Recovery can be with a suctionpipette, for example, to allow further studies (i.e. adoptive transferinto small animals, further testing in a variety of settings, such assingle cell microarray gene expression profiling etc.).

As noted above, in some embodiments of the invention, the method is usedto observe a viscoelastic property of a live cell in an aqueous medium.Optionally, the method is used to observe a population of live cells,for example to observe resting and dynamic responses to stimuli in apopulation of live cells. In certain embodiments of the invention,resting and/or dynamic responses of a plurality of cells in a populationof live cells can be measured simultaneously. Typically in thesemethods, the property is observed in response to the cell's exposure toa stimulus such as the magnetic field applied to the cell and/or acomposition introduced into the cell's media. Optionally the methodsfurther comprise removing the cell from the observation chamber andmanipulating the cell for a further analysis. In certain embodiments ofthe invention, the method is used to obtain information comprising acell specific profile of a live cell in an aqueous medium and to thenstore this information in the memory storage element.

In some embodiments of the invention, cells can be arrayed for moreuniform, higher density, and higher throughput analysis (e.g. byphotoresist deposition processes known in the art) with “holes” (e.g.nanowells or microwells) of an appropriate size (see, e.g. FIGS. 13A and13B). In this context, microreflector placement on the surface of cellscan be guided/enhanced by manufacture that is less than the size of awell, creating a sort of “piston-like” action that eliminates manyissues related to exact reflector placement on the surface of eachindividual cell.

Embodiments of the invention also include a reflective microparticlecomprising a gradient index (GRIN) spherical lens. Typically thismicroparticle is coupled to a flexible tether and/or an optical fiberand/or is operatively coupled to an endoscope. In certain embodiments ofthe invention, this microparticle comprises a plurality of materiallayers, wherein refractive indices of the material layers decrease fromthe center of the microparticle. Further aspects, elements, andprocesses associated with embodiments of the invention are disclosedbelow.

ILLUSTRATIVE METHODOLOGICAL EMBODIMENTS OF THE INVENTION

The invention disclosed herein has a number of methodologicalembodiments. Typical embodiments of the invention comprise a method fordetermining one or more characteristics of one or more cells includingthe steps of directing an incident beam of light on the cell, whereinthe cell comprises a subject micromirror positioned on the cell surface;and then detecting a beam of light reflected from the micromirror, todetect a movement of the membrane of the cell. In some embodiments, themembrane movement detected is in response to an external stimulus (e.g.a factor delivered into the liquid medium environment in which the cellis disposed). In other embodiments, the membrane movement is in responseto an internal stimulus.

Embodiments of the invention further include a method for determining acell characteristic by detecting membrane movement in a plurality ofcells. The method comprises contacting each cell of a plurality of cellswith a micromirror probe and then detecting movement of the probes (e.g.in response to various forces and/or stimuli). In some of theseembodiments, a plurality of tests (e.g. in the form of an array) isperformed. In some embodiments of the invention, the method furthercomprises allowing a signal resulting from membrane movement of a firstcell in a plurality of cells in response to a stimulus to be transmittedto a second cell in the plurality of cells; and then detecting movementof a membrane in the second cell in response to the transmitted signal.

In embodiments of the invention where a cell is in a liquid medium, anexternal stimulus can added to the medium, and the response of the cell,as detected by membrane movement, to the external stimulus is monitored.In other embodiments, where the external stimulus is a condition such asheat, cold, radiation, etc., the condition of the cell's environment isadjusted. The methods are useful for detecting changes in a cell evenbefore the change is detectable visually. For example, a cancerous cellis detected even before the cell undergoes characteristic morphologicalchanges that are visible when viewed under a microscope (e.g., by aclinician). In these embodiments, a cancerous state in a cell isdetected in vitro in a biological sample (e.g., a cervical swab, orother tissue biopsy sample) by detecting cell membrane movement, andcomparing the movement of the cell membrane with the cell membranemovement characteristic of a normal cell of the same cell type. In thismanner, cancerous cells can be detected at a much earlier stage thanwith means currently available to the clinician.

Another embodiment of the invention is a method of identifying an agentthat affects a biological activity of a cell, the method generallyinvolving contacting a cell with a test agent, and determining theeffect, if any, of the agent on the biological activity of the cell,wherein the determining comprises detecting cell membrane movement.Another embodiment of the invention is a method of identifying acharacteristic of a test cell, the method generally involving:determining a cell characteristic profile of the test cell to generate atest profile, wherein the cell characteristic profile comprises cellmembrane movement data and at least one additional cell parameter; andcomparing the test profile with a reference profile in a database ofprofiles, wherein this comparison can be used to identify a referenceprofile that is similar or substantially identical to the test profile,and wherein the reference profile identifies the cell characteristic. Incertain embodiments of the invention, the reference profile indicatesthat the test cell is abnormal (e.g. a cell in a tissue biopsy from apatient suspected of having a cancer). Another embodiment of theinvention is a method for screening for the presence of a biologicallyactive agent in a sample (e.g. a ligand that binds to a receptorexpressed on the surface of a cell), the method generally involving:contacting a cell with a test sample suspected of containing an agent;and determining the effect, if any, of the test sample on the cellmembrane movement of the cell.

As noted above, in typical embodiments of the invention, membranemovement is detected using an Michelson interferometer. This membranemovement is used as a read-out for a cell's response to a biochemicalevent and/or the physiological status of a cell and/or the cell type.Embodiments of this method allow for real-time monitoring of a cell'sresponse to an internal or external stimulus. Embodiments of theinvention include methods for determining a characteristic of a cell,such as cell type, cellular response to a biochemical event, andbiological state. These methods typically involve detecting membranemovement in a cell and then using the movement detected by this methodto obtain information on a cell characteristic. The methods of theinvention are useful for applications such as the screening ofbiologically active (e.g. therapeutic) agents as well as diagnosticapplications such as identifying a cell having one or morecharacteristics associated with a pathological condition such as cancer.Certain embodiments of the invention include methodological steps thatemploy one or more databases of cell characteristics, as known in theart and/or as determined using the disclosed methods and systems.

As noted above, embodiments of the invention provide methods ofdetermining a characteristic of a cell, typically a living cell. Themethod generally involves detecting movement of a cell membrane, andrelating the movement to a cell characteristic. Membrane movement isdetected using a system that includes at least a membrane movementresponsive element (e.g. a microsphere) and a detection element fordetecting a signal generated by the responsive element (e.g. aninterferometer). The system may further include other elements known inthe art, such as a data storage means for storing the signal; and a dataprocessing means, for converting the signal to various formats, forcomparing the signal to other stored signals, etc.

A typical method for observing membrane movement includes contacting themembrane with a micromirror element that responds to membrane movementand provides a detectable signal in response to-membrane movement. Thesignal is then transmitted or detected by a detection element. Thesignal detected by the detection element is transmitted to a dataprocessing and storage means, e.g. a computer system. The system mayalso include an element for transmitting a signal to a cell membrane.Membrane movement includes, but is not limited to, lateral movement,stretching, contracting, and the like.

Embodiments of the invention can be used to detect membrane movement ina variety of cells (including naturally-occurring cells and artificialcells) as well as subcellular organelles. Cells that can be examined byembodiments of the invention include eukaryotic cells; prokaryoticcells; and artificial cells. Eukaryotic cells include mammalian cells,reptilian cells, amphibian cells, yeast cells, plant cells, protozoancells, algae, and the like. Subcellular organelles include the nucleus,mitochondria, Golgi apparatus, vacuoles, and the like. Prokaryotic cellsinclude bacterial cells (e.g., eubacteria), and archaebacterial cells.Cells and cellular environments that can be examined by embodiments ofthe invention include cells in vitro and in vivo, e.g., isolated cellsin vitro; cells in colonies in vitro; cells in tissues in vitro; singlecells in vivo; cells in tissues in vivo; unicellular organisms; cells ofmulticellular organisms; and the like.

Embodiments of the invention can be used to detect membrane movementassociated with biochemical events. Biochemical events include internalstimuli; external stimuli; gene expression; and the like. Movement ofthe membrane is detected in response to a change in physiologicalconditions in the cell or organelle. Changes in physiological status aregenerally in response to an internal or an external signal. External andinternal signals (stimuli) include, but are not limited to, infection ofa cell by a microorganism, including, but not limited to, a bacterium(e.g., Mycobacterium spp., Shigella, Chlamydia, and the like), aprotozoan (e.g., Trypanosoma spp., Plasmodium spp., Toxoplasma spp., andthe like), a fungus, a yeast (e.g., Candida spp.), or a virus (includingviruses that infect mammalian cells, such as human immunodeficiencyvirus, foot and mouth disease virus, Epstein-Barr virus, and the like;viruses that infect plant cells; etc.); change in pH of the medium inwhich a cell is maintained or a change in internal pH; excessive heatrelative to the normal range for the cell or the multicellular organism;excessive cold relative to the normal range for the cell or themulticellular organism; an effector molecule such as a hormone, acytokine, a chemokine, a neurotransmitter; an ingested or applied drug;a ligand for a cell-surface receptor; a ligand for a receptor thatexists internally in a cell, e.g., a nuclear receptor; hypoxia; a changein cyoskeleton structure; light; dark; mitogens, including, but notlimited to, lipopolysaccharide (LPS), pokeweed mitogen; stress;antigens; sleep pattern (e.g., sleep deprivation, alteration in sleeppattern, and the like); an apoptosis-inducing signal; electrical charge(e.g., a voltage signal); ion concentration of the medium in which acell is maintained, or an internal ion concentration, exemplary ionsincluding sodium ions, potassium ions, chloride ions, calcium ions, andthe like; presence or absence of a nutrient; metal ions; a transcriptionfactor; a tumor suppressor; cell-cell contact; adhesion to a surface;peptide aptamers; RNA aptamers; intrabodies; and the like.

Responses to internal stimuli that can be observed using embodiments ofthe method can include the expression of a gene, and/or production of agene product. Production of a gene product includes expression of anendogenous gene, as well as the expression of an introduced nucleicacid. In some embodiments, a nucleic acid comprising a nucleotidesequence encoding a protein of interest is introduced into a cell,generating a genetically modified cell, the nucleic acid is expressed,the protein is produced in the genetically modified cell, and theresponse of the genetically modified cell to the protein is detected. Inone embodiment, the nucleic acid encodes a nucleic acid that affectstranscription of a gene. Such nucleic acids include antisense nucleicacids, ribozymes, and inhibitory RNA (RNAi) (including double strandedRNAi).

Embodiments of the invention can be used to detect the presence and/orlevel of one or more proteins of interest. In some embodiments, theprotein of interest is an exogenous protein, e.g., a protein that thecell does not normally produce because the cell does not possess anucleic acid encoding such a protein (or possesses a nucleic acid thatdoes not express that protein). In other embodiments, the protein ofinterest is one that a normal cell of the same cell type would normallyproduce, but that the cell cannot produce because of some defect in thecell. For example, the cell's genome may contain a mutation in thecoding region and/or regulatory region of a gene such that a givenprotein is not produced. Alternatively, the cell's genome may contain adefect in a coding region and/or regulatory region in a gene other thanthe gene encoding the protein of interest, and introduction of thenucleic acid encoding the protein of interest circumvents the defect.For example, the cell may contain a mutation in a gene encoding atranscription factor necessary for production of the protein ofinterest, such that the transcription factor is either not produced oris produced in an inactive form, and a nucleic acid is introduced whichencodes the protein of interest under control of a promoter notregulated by the absent or defective transcription factor. In otherembodiments, the protein of interest is a dominant negative mutant,e.g., a protein that, when produced, prevents a counterpart normalprotein that is produced by the cell from functioning, or reduces thefunction of the normal protein. In other embodiments, the protein is anendogenous protein that the cell produces at low levels, andintroduction of a nucleic acid encoding the protein results inover-production of the protein. In these embodiments, the coding regionof the protein of interest is operably linked to a promoter element.Constitutive promoters that are active in most eukaryotic cell are wellknown in the art, and include, but are not limited to, humancytomegalovirus immediate early promoter, adenovirus major latepromoter, an SV40 virus promoter, a Rous sarcoma virus promoter, and amurine 3-phosphoglycerate kinase promoter. The nucleotide sequenceencoding the protein of interest can also be under transcriptionalcontrol of an inducible promoter, e.g., a promoter that can be activatedby an inducer; a repressible promoter, or a developmentally regulatedpromoter. For example, where the nucleotide sequence encoding theprotein of interest can be under transcriptional control of an induciblepromoter, an external signal includes an inducer of an induciblepromoter. Inducible promoters, and their inducers, are well known in theart, and include, but are not limited to, cold-inducible promoters;heat-inducible promoters; metal ion inducible promoters; atetracycline-inducible promoter; a radiation-inducible promoter; adrug-inducible promoter; a hormone-inducible promoter; and the like. Inthese embodiments, the nucleic acid is introduced into the cell, and theinducing agent is added to the medium, or the cell is exposed to theinducing condition which results in expression of that gene (e.g., achemical inducer, heat, cold, radiation, etc.).

The methods of the invention find use in a variety of applications,including drug screening, detection assays, and diagnostic assays. Theinvention provides screening assays for identifying agents that affect acell process and/or physiological status. The invention further providesassays for detecting the presence of an analyte in a test sample. Themethods generally involve contacting a cell with a test agent; anddetecting any change in cell membrane movement in response to the testagent. For example, an agent that inhibits mitosis, and which thereforemay be of use in treating cancer, is identified by monitoring cellmembrane movement that is characteristic of a cell undergoing mitosis.

The terms “candidate agent,” “agent,” “substance,” and “compound” areused interchangeably herein. Test agents encompass numerous chemicalclasses, typically synthetic, semi-synthetic, or naturally-occurringinorganic or organic molecules. Test agents may be small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Test agents may comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andmay include at least an amine, carbonyl, hydroxyl or carboxyl group, andmay contain at least two of the functional chemical groups. The testagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Test agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Test agents include those found in large libraries of synthetic ornatural compounds. For example, synthetic compound libraries arecommercially available from Maybridge Chemical Co. (Trevillet, Cornwall,UK), ComGenex (South San Francisco, Calif.), and MicroSource (NewMilford, Conn.). A chemical library is available from Aldrich(Milwaukee, Wis.). Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available fromPan Labs (Bothell, Wash.) or are readily producible. Additionally,natural or synthetically produced libraries and compounds are readilymodified through conventional chemical, physical and biochemical means,and may be used to produce combinatorial libraries. Libraries of testagents also include cDNA libraries, e.g., expression libraries from agiven cell type, from a cell in response to an agent, from a cell of agiven physiological status (e.g., a cancerous cell), and the like. Inaddition, known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs. Newpotential therapeutic agents may also be created using methods such asrational drug design or computer modeling.

Assays of the invention include controls, where suitable controlsinclude a sample (e.g., a cell sample) in the absence of the test agentor other condition. Generally, a plurality of assay mixtures is run inparallel with different agent concentrations to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

Agents that have an effect in an assay method of the invention may befurther tested for cytotoxicity, bioavailability, and the like, usingwell known assays. Agents that have an effect in an assay method of theinvention may be subjected to directed or random and/or directedchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs. Such structuralanalogs include those that increase bioavailability, and/or reducedcytotoxicity. Those skilled in the art can readily envision and generatea wide variety of structural analogs, and test them for desiredproperties such as increased bioavailability and/or reduced cytotoxicityand/or ability to cross the blood-brain barrier. The components of theassay mixture are typically added in any order that provides for therequisite binding or other activity. Incubations are performed at anysuitable temperature, typically between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hour will be sufficient.

In many embodiments, an array of isolated cells or colonies of cells isused, wherein each isolated cell or colony of cells is contacted with atest agent, and the effect on the cells is determined by contacting thecells or cell colony with a micromirror and examining one or morecharacteristics of the cell(s) via interferometry. In some embodiments,a cellular array is addressable, such that the identity of each isolatedcell/cell colony is known and can be matched to the cell's reaction tothe test agent. For example, cells are deposited on discrete regions(e.g., microwells) of a solid substrate, and each microwell contains aunique identifier that corresponds to the identity of the cells in themicrowell. Alternatively, the cells themselves are coated with at leastone optically interrogatable material, such as a bioluminescentcompound, a chemiluminescent compound, a chromophore, a fluorophore,etc. (see, e.g., U.S. Pat. No. 6,377,721, the contents of which areincorporated by reference).

The invention further provides assays for detecting the presence of ananalyte in a test sample. The methods generally involve contacting acell with a test sample; and detecting any change in cell membranemovement in response to the test sample. Such a screening assay isuseful to detect the presence in a sample of an biologically activeagent suspected to exist in the sample, e.g., a subject screening assaycan be used to detect the presence in a sample of a toxin or a toxicbacterium, e.g., an environmental agent (e.g., a pesticide, anherbicide, an environmental toxin, and the like), an agent of chemicalor biological warfare (e.g., nerve gas, anthrax, etc.). Assays of theinvention include controls, where suitable controls include a sample(e.g., a cell sample) in the absence of the test sample. Generally aplurality of assay mixtures is run in parallel with different knownconcentrations of the agent being detected to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection. The assay methods provide forqualitative (e.g., presence or absence), semi-quantitative, andquantitative detection of analyte. Where the methods are quantitative,the response of a cell membrane to a test sample is compared to astandard curve obtained using known concentrations of the analyte, andthe presence and concentration of the analyte are determined.

As an example, the methods of the invention are useful for detecting thepresence in a tissue or a biological sample of a cell that is abnormal.Because the cell membrane movement is characteristic for a given celltype, and also for cancerous cells of a given cell type, determinationof the cell membrane movement of a given cell provides information as towhether the cell is undergoing mitosis at a rate characteristic for thecell type, or is dividing in an uncontrolled manner, e.g., is cancerous.The methods are also useful for providing the cell type of the cancerouscell. The methods are further useful for staging the cancer. In thiscontext, the invention further provides a method of treating a diseaseor disorder. The methods generally involve identifying a characteristicof a cell, which characteristic indicates that the cell is abnormal; andrecommending and/or selecting a treatment regimen appropriate to theabnormality. For example, where a cell in a tissue biopsy is determinedto be a cancerous cell, a treatment regimen appropriate to theparticular type of cancer is recommended. In some embodiments, asdiscussed above, the methods provide for staging of the cancer. A courseof chemotherapy or radiation therapy appropriate to the stage of thecancer is then recommended.

Embodiments of the invention useful for identifying a characteristic ofa test cell can be coupled to computer systems and databases. Methodsfor identifying a characteristic of a test cell generally involvedetermining a cell characteristic profile of the test cell to generate atest profile, and comparing the test profile with a reference profile ina subject database. Such methods further include the generation of alibrary of profiles (e.g. one grouped according to specificphysiological conditions associated with various profiles) as well ascomparisons of a test profile to profiles in a library of profiles. Suchcomparisons can use software processes known in the art to provide thebest match, e.g., to identify a reference profile that is substantiallyidentical to the test profile. The reference profile can then be used tocorrelate one or more characteristics of the test cell (e.g.disregulated cell growth).

The cell characteristic profiles can be compiled in a database, asdescribed above, and the information in the database is used to comparethe profile of a test cell to a reference profile in the database. Thecomparison can be made by trained personnel (e.g., a clinician, atechnician, etc.), or can be made by a computer or other machine. Thesubject diagnostic assays are useful for identifying any type ofabnormal cell, for example, diagnostic assays of the invention areuseful for identifying cancerous cells in a biological sample, e.g., abiopsy, as well as in an individual in vivo.

The data obtained from analysis of various cell types under variousphysiological conditions and in various physiological states can becompiled in a database in order to, for example, train neural networksfor independent detection of cell types and physiological status ofcells. The cell characteristic profiles are obtained as described above,and the neural network is trained to recognize cells of various celltypes, cells in various physiological states, and cells responding tovarious stimuli. The neural network is useful for identifying cancerouscells, pre-cancerous cells, and cells in other pathological conditions.

Illustrative Systems and Materials Used with Embodiments of theInvention

Embodiments of the invention include systems for determining acharacteristic of a cell. A typical system generally includes at least amembrane movement responsive element (e.g. a micromirror); and adetection element for detecting a signal generated by the responsiveelement (e.g. an interferometer) as well as a data storage andprocessing means, for storing the signal, for converting the signal tovarious formats, for comparing the signal to other stored signals, etc.Data storage and processing means are computer-based systems, asdescribed below. In some embodiments, where the detection systeminvolves use of a micromirror attached to a cell surface, a subjectsystem includes a light source (e.g., a LED), a sensor, such as a CCD,and data storage and processing means.

Interferometry Systems

Interferometry is the technique of diagnosing the properties of two ormore optical waves by studying the pattern of interference created bytheir superposition. The Michelson interferometer is a commonconfiguration for optical interferometric device. In such devices, aninterference pattern is produced by splitting a beam of light into twopaths, bouncing the beams back and recombining them. Art in the field ofinterferometry teaches that different paths may be of different lengthsor be composed of different materials to create alternating interferencefringes on a detector. A variety of interferometers and interferometrysystems known in the art can be used and/or adapted for use withembodiments of the invention. Typical interferometers and interferometrysystems include those disclosed in Basics of Interferometry, SecondEdition by P. Hariharan (2006); Optical Interferometry, Second Editionby P. Hariharan (2003); Two-Dimensional Phase Unwrapping: Theory,Algorithms, and Software by Dennis C. Ghiglia and Mark D. Pritt; U.S.Pat. Nos. 7,505,863; 7,212,356; 6,624,893; 6,459,489; 7,522,282; and7,492,462 and U.S. Patent Application Nos. 20070076208; 20080218999;20030004412; and 20050190372, the contents of each of which isincorporated by reference herein.

Probes

Embodiments of the invention include a probe that is operatively coupledto the membrane of one or more cells (e.g. a micromirror such as themicrometer sized elemental nickel microspheres disclosed in the Examplesbelow). The probe typically is of a radius such that movement of amembrane of a cell that correlates with one or more physiologicalphenomena can be readily detected (e.g. spherical magneticmicroparticles having an average diameter of between 1 μm and 15 μm).

As noted above, typical embodiments of the invention provide a methodfor determining a cell characteristic, involving monitoring the movementof a mirror attached to the cell surface. Use of a mirror attached to acell surface is particularly useful for analyzing soft cells, such asmammalian cells. For example, where the cell characteristic isdetermined by monitoring cell membrane movement, cell membrane movementis analyzed by detecting movement of a mirror (also referred to hereinas a “reflector,” a “micromirror”) attached to the surface of the cell.As used herein, the term “reflector” is used to denote a body whichreflects a portion of the electromagnetic radiation incident on thebody. An incident beam of light is reflected by the mirror. Movement ofthe mirror is detected by a sensor which detects the reflected beam oflight.

Optionally such micromirrors further comprise a cell attachment surface,wherein the surface area of the micromirror is in a range of from about25 nm² to about 75μ². In some embodiments, the reflector surface cancomprise a diffraction grating. In some embodiments, the cell attachmentsurface of the micromirror comprises a cell attachment moietyimmobilized on the cell attachment surface. Typical cell moietiesinclude, e.g., an antibody, a polypeptide, an integrin, a virusattachment protein, a carbohydrate, and a ligand for a cell surfacereceptor.

Embodiments of the invention can be used to detect vertical or lateralmovement of a cell membrane of from about 0.1 nm to about 500 nm, e.g.,from about 0.1 nm to about 1 nm, from about 1 nm to about 5 nm, e.g.,from about 5 nm to about 10 nm, from about 10 nm to about 50 nm, fromabout 50 nm to about 100 nm, from about 100 nm to about 250 nm, or fromabout 250 nm to about 500 nm. Embodiments of the invention can detectmovement at a frequency of from about 1 Hz to about 10 KHz, e.g., fromabout 1 Hz to about 10 Hz, from about 10 Hz to about 50 Hz, from about50 Hz to about 100 Hz, from about 100 Hz to about 500 Hz, from about 500Hz to about 1 kHz, or from about 1 kHz to about 10 kHz. The data can beexpressed as vertical (or lateral) displacement versus time. In certainembodiments, the data are treated with a Fourier Transform (FT) togenerate an amplitude spectrum. In embodiments of the invention,measurements of membrane movement can made at regular intervals (e.g.,every 1, 5, 10, 30 seconds, or 60 seconds, and/or 2, 3, 4, 5, 6, 7, 8, 9or 10 minutes, 15 minutes, etc.); or continuously. Alternatively,measurements of membrane movement are made at a single time point (e.g.a predetermined or random time point).

Where the response of a cell to an external stimulus is analyzed invitro, the external stimulus is added to the cell medium, as describedabove. In some embodiments, an external stimulus is added to the mediumin which a cell is being analyzed (e.g., cell culture medium, bodilyfluid, etc.). In other embodiments, the external stimulus is deliveredby the probe itself (e.g. a probe coated with an agent of interest suchas a biologically active agent). In these embodiments, the probe can befunctionalized or adapted to deliver the signal. In these embodiments, astimulus is attached to the probe. For example, a drug, a hormone, anucleic acid, or other signal is attached to the probe, and when theprobe contacts the membrane, the stimulus is delivered to the cell. Thestimulus can be linked to the probe either covalently or non-covalently.

Alternatively, the probe is fitted with an element that delivers thestimulus. For example, a single or multiwall carbon nanotube is attachedto the probe, and an external stimulus (e.g., an agent in a liquidformulation) is delivered into the cell (e.g., into the cytoplasm orinto the nucleus) by the nanotube. In these embodiments, the stimulus islinked (covalently or non-covalently) to the nanotube. In someembodiments, the stimulus is attached to the nanotube via a linker whichis proteolytically cleaved by an intracellular enzyme. In someembodiments, an element for delivering an electrical signal will beattached to the probe, or is positioned adjacent to the probe, such thatan electrical signal is delivered to the membrane. In some embodiments,a stimulus that is delivered to the cell membrane is a programmable orpre-recorded pattern that is stored in a data storage medium. Forexample, a stimulus is delivered to the cell at regular intervals (e.g.,to mimic a Circadian rhythm). As another example, a stimulus isdelivered to a cell to stimulate entry into the cell cycle at aparticular time point, which is pre-recorded.

In some embodiments, movement of a membrane of a first cell is convertedinto a signal, and transmitted to the membrane of a second cell. In someof these embodiments, the first cell is physically separated from thesecond cell, e.g., the first cell and the second cell are in separatewells of a multi-well plate. In other embodiments, the first cell andthe second cell are in the same tissue or colony, but are separated fromone another by other cells and/or extracellular matrices. In otherembodiments, the first cell and the second cell are in direct contactwith one another. Any movement in a membrane of the second cell inresponse to the signal transmitted from the first cell can betransmitted to a third cell, and so on.

In some embodiments of the invention, a single probe is in contact witha cell. In other embodiments, two, three, four, or more, individualprobes are in contact with a membrane of a cell. For example, individualprobes are in contact with different areas of a cell membrane. Anexample of a situation in which use of multiple probes is useful is inanalyzing cells in which different areas of the cell responddifferentially to a given stimulus. Non-limiting examples of such cellsare polarized cells (e.g., columnar epithelial cells lining thegastrointestinal tract); and cells that have processes that extend fromthe cell body (e.g., neuron, axons, dendritic cells, etc.).

Embodiments of the invention include the steps of processing dataobtained by a detecting device. For example, data transmitted by theposition-sensitive detector to the data processor can be formatted inseveral different ways. In one embodiment, the data can be processedusing a Fourier Transformation analysis such as Fourier TransformationFiltering (FTF). The data can also be converted into audio or colorformat. Conversion into audio format is accomplished using standardaudio conversion software, and allows a qualitative measure of thephysiological status of a cell. In addition, rather than representingthe amplitudes as peaks, color intensities can be used to represent peakintensities, and a plot of each short FTF versus time can be made,resulting in a sonogram. For example, the data can be converted into ausable format in real time using Fourier Transformation Filtering, AudioFiles, Color Spectra, and the like.

In embodiments of the invention, the movement of an entire cell can bemonitored in vitro. Movement of a cell includes lateral movement andvertical movement. The movement of a cell is in some embodimentsmonitored in an in vitro culture, wherein the cells are detached fromone another. In some embodiments, movement of a cell in an in vitroculture system involves monitoring movement of a cell across a monolayerof adherent cells. In other embodiments, movement of a cell in an invitro culture system involves monitoring movement of a cell in a tissue.In still other embodiments, movement of a cell in an in vitro culturesystem involves monitoring adhesion of a first cell to a second cellthat comprises on its cell surface a receptor for a ligand present onthe surface of the first cell, or that comprises a co-receptor for areceptor present on the surface of the first cell. As one non-limitingexample, metastasis of a tumor cell can be monitored in in vitro cellculture assay. As another non-limiting example, leukocyte homing andextravasation can be monitored in an in vitro assay.

In typical embodiments of the invention, light, e.g., light from a LED,a laser beam or any one of the wide variety of optical sources known inthe art, is directed onto a mirror attached to the surface of a cell,and a position of reflected light is detected by a sensor. For example,a first position of optical radiation reflected by a mirror attached toa cell surface is detected at a first time (e.g. by a first photographor the like); and a second position of optical radiation reflected bythe mirror is detected at a second time (e.g. by a second photograph orthe like). Where the second position differs substantially from thefirst position indicates movement of the cell membrane (includingmovement of the entire cell).

Mirrors suitable for attachment to the cell surface are any of a varietyof shapes, e.g., circular, oval, flat cylindrical, square, ellipsoid,rectangular, an irregular shape, etc. In many embodiments, a subjectmicromirror is generally disc shaped. Typically, the surface area of themirror is less than about 50%, less than about 40%, less than about 30%,less than about 20%, less than about 10%, less than about 5%, less thanabout 2%, or less than about 1% of the surface area of the cell. Forexample, in general, the surface area of one group of micromirrorembodiments is in a range of from about 75 nm² to about 25 μm², e.g.,from about 75 nm² to about 100 nm², from about 100 nm² to about 150 nm²,from about 150 nm² to about 200 nm², from about 200 nm² to about 500nm², from about 500 nm² to about 750 nm², from about 750 nm² to about 1μm², from about 1 μm² to about 2 μm², from about 2 μm² to about 5 μm²,from about 5 μm² to about 10 μm², from about 10 μm² to about 15 μm² fromabout 15 μm² to about 20 μm², or from about 20 μm² to about 25 μm².

The diameter of a typical mammalian cell ranges from about 3 μm to about11 μm. In certain embodiments of the invention where the mirror isspherical, and the cell is a mammalian cell, the mirror can have adiameter in the range of from about 10 nm to 30 μm, e.g., from about 10nm to about 50 nm, from about 50 nm to about 100 nm, from about 100 nmto about 250 nm, from about 250 nm to about 500 nm, from about 500 nm toabout 750 nm, from about 750 nm to about 1 μm, from about 1 μm to about2 μm, from about 2 μm to about 5 μm, from about 5 μm to about 10 μm,from about 10 μm to about 15 μm, from about 15 μm to about 20 μm, fromabout 20 μm to about 25 μm, or from about 25 μm to about 30 μm etc. Theinstant disclosure provides evidence that micromirrors used to studylive mammalian cells disposed within a observation chamber of aninterferometer sample assembly optimally have a diameter in the rangewithin about 0.5 μm to 30 μm (e.g. 5 μm to 15 μm).

The reflector surface of a subject mirror comprises any of a variety ofmaterials. Suitable materials for the reflector surface include, but arenot limited to nickel and other reflective metals and metal alloys,silicon dioxide, polydimethylsiloxane (PDMS), silicon nitride (SiNx),and the like. In some embodiments, a subject mirror comprisesbiodegradable materials. The reflector surface of a subject mirror willin some embodiments comprise an optical grating texture (a “diffractiongrating”) that diffracts light. The diffraction grating is in any of avariety of patterns, e.g., a linear array, a radial array, a spiralarray, and the like. In addition, the pitch of the grating can vary.See, e.g., “Diffraction Grating” 5th edition, C. Palmer (2004)Spectra-Physics, Inc. For example, the pitch can vary from about 0.1 μmto about 10 nm, e.g., from about 0.1 μm to about 0.5 μm, from about 0.5μm to about 1 μm, from about 1 μm to about 10 μm, from about 10 μm toabout 50 μm, from about 50 μm to about 100 μm, from about 100 μm toabout 500 μm, from about 500 μm to about 1 nm, from about 1 nm to about5 nm, or from about 5 nm to about 10 nm. In some embodiments, thegrating pattern provides information as to the identity of theattachment molecule(s) on the attachment surface of the mirror. Thus, inthese embodiments, the diffraction grating pattern provides a code as tothe identity of the attachment molecule or combination of attachmentmolecules on the attachment surface.

Attachment Moieties

The mirror can be attached to the surface of a deformable compositionsuch as a cell membrane by any of a number of interactions, includingelectrostatic interactions, steric stabilization, van der Waals forces,gravitational forces, frictional forces, covalent linkage, and the like.In some embodiments, an attachment moiety is attached to (immobilizedon) an attachment surface of the mirror, where the attachment moietyprovides for attachment of the mirror to the cell surface. In someembodiments, an attachment moiety is synthesized directly on theattachment surface of the mirror. See, e.g., U.S. Pat. No. 6,630,308. Inother embodiments, a preformed attachment moiety is attached to(immobilized on) the attachment surface by chemical coupling, adsorptionor other means. A large number of immobilization techniques have beenused and are well known in the fields of solid phase immunoassays,nucleic acid hybridization assays and immobilized enzymes. See, forexample, Hermanson, Greg, T. Bioconjugate Techniques. Academic Press,New York. 1995, 785 pp; Hermanson, G. T., Mallia, A. K. & Smith, P. K.Immobilized Affinity Ligand Techniques. Academic Press, New York, (1992)(Chapter 5); and Avidin-Biotin Chemistry: A Handbook. D. Savage, G.Mattson, S. Desai, G. Nielander, S. Morgansen & E. Conklin, PierceChemical Company, Rockford Ill., 1992, 467 pp; Protein Immobilization,Fundamentals & Applications, R. F. Taylor, ed. (1991) (chapter 8).

Attachment moieties include a wide variety of biomolecules, including,but not limited to, nucleic acids, including DNA, RNA, oligonucleotides;proteins (including phosphoproteins, lipoproteins, glycoproteins, andthe like), peptides, lipids, fatty acids; polysaccharides,oligosaccharides; organic polymers; any of a wide variety of organicmolecules which include one or more moieties for binding to a cellsurface biomolecule; antibodies; ligands (e.g., agonists for a cellsurface receptor, an antagonist for a cell surface receptor);microorganisms; receptors; antibiotics; test compounds (e.g., compoundsproduced by combinatorial chemistry); bacteria; viruses; and plant andanimal cells and organelles or fractions thereof.

An attachment moiety is bound to (immobilized onto) the attachmentsurface of the mirror. The mirror is bound to the cell surface. The term“bind” includes any physical attachment or close association, which maybe permanent or temporary. Generally, an interaction of hydrogenbonding, hydrophobic forces, van der Waals forces, covalent bonding,etc. facilitates physical attachment between the attachment moiety andthe attachment surface. Generally, an interaction of hydrogen bonding,hydrophobic forces, van der Waals forces, etc. facilitates physicalassociation between an attachment moiety and a cell surface molecule.

The attachment surface of the mirror comprises any material onto whichan attachment moiety can be immobilized, or that can be derivatized orotherwise processed such that an attachment moiety can be immobilizedonto the attachment surface. Suitable attachment surface materialsinclude, but are not limited to, polymers, plastics, resins,polysaccharides, silica or silica-based materials, carbon, metals,inorganic glasses, membranes, particles, gels, functionalized glass,germanium, silicon, GaAs, GaP, SiO2, SiN4 modified silicon,polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene,polycarbonate, flat glass, or single-crystal silicon,polytetrafluorethylene, polystyrene, gallium arsenide, or combinationsthereof. Suitable polymers include, but are not limited to,(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, and polymerized Langmuir-Blodgett film.

The attachment surface of a subject mirror is in many embodimentsfunctionalized to include one or more attachment sites for attaching tothe cell surface, or for attaching an attachment moiety. Suitablefunctional groups include, but are not limited to, sulfhydryl groups,and the like. The attachment surface may be coated with a material thatfacilitates attachment of an attachment moiety. Some solid phasesurfaces may be used directly to immobilize an attachment moiety, othersmust be modified to allow such attachments. For example, antibodies andmany other proteins will adhere to clean polystyrene surfaces.Polystyrene, either in the form of microtiter plates or beads, have beenmodified to bind nucleic acids, proteins, and polysaccharides usingtechniques that are well known. TEFLON surfaces will bind proteins orother macromolecules that have been suitably fluorinated (see, e.g.,U.S. Pat. No. 5,270,193) and will also bind fluorinated surfactants,which may render the surface hydrophilic, or positively or negativelycharged. Glass, including controlled pore glass, may be modified toallow covalent attachment of antibodies, antigens or nucleic acids.Plastic or glass surfaces may be modified non-specifically using coronaplasma discharge or electron beam radiation and may then be coated witha variety of coatings or adhesives to which an attachment moiety may beattached. More specific covalent attachment of proteins, nucleic acidsor carbohydrates may be achieved by a variety of modifications whichattach reactive groups to polystyrene or acrylic surfaces, which groups,with or without extending linkers, will then couple under mildconditions to the biopolymers.

In addition to methods by which an attachment moiety is immobilized on asolid surface, general methods exist for immobilizing members of a classof attachment moiety. For example, protein A or protein G may beimmobilized and used to subsequently bind specific antibodies which inturn will bind specific ligands. A more general approach is built aroundthe strong and specific reaction between other ligands and receptorssuch as avidin and biotin. Avidin may be immobilized onto the attachmentsurface, and used to bind antibodies or other biomolecules to whichbiotin has been covalently linked. This allows the production ofsurfaces to which a very wide variety of reactants can be readily andquickly attached (see, e.g. Savage et al., Avidin-Biotin Chemistry: AHandbook. Pierce Chemical Company, 1992).

In some embodiments, the attachment moiety is a first member of aspecific binding pair, where the second member of the specific bindingpair is displayed on the cell surface. Non-limiting examples of specificbinding pairs include selectin/selectin ligand; viral antigen/cellsurface receptor; antibody/antigen; receptor/ligand; extracellularmatrix/integrin; and the like. Thus, e.g., the first member of aspecific binding pair is one or more of the following: an antibody (oran epitope-binding fragment thereof) specific for an epitope displayedon a cell surface; a ligand for a cell surface receptor; a portion of anextracellular matrix molecule that is bound by a cell surface receptor;a carbohydrate moiety that is recognized by a cell surface selectin; andthe like.

Viral antigens that are suitable for attachment to the attachmentsurface of a subject mirror include, but are not limited to, any viralattachment protein, e.g., a viral env protein, a viral spike protein, aviral fusion protein, a viral capsid protein, and the like, including,e.g., human immunodeficiency virus (HIV) gp160 and gp120; humanrhinovirus 14; tick-borne encephalitis virus E protein; influenza virushemagglutinin; respiratory syncytial virus fusion protein F; adenovirusfiber protein; reovirus attachment protein .sigma.1; SARS coronavirusS(S1) protein; herpes simplex virus-1 glycoprotein D; poliovirus capsidshell (VP1, VP2, VP3); and the like. Viral entry proteins are welldescribed in the literature. See, e.g., Dimitrov (2004) Nature2:109-122. In addition, suitable agonists and antagonists that bind acell surface receptor include, but are not limited to, hormones;neurotransmitters; cytokines; chemokines; pharmaceutical agents;derivatives of any of the foregoing that have altered propertiescompared to a naturally-occurring agonist or antagonist; and the like.

In some embodiments, the attachment moiety is specific to a cell type.In other embodiments, the attachment moiety provides attachment to awide variety of cells. Non-limiting examples of attachment moieties thatare specific to particular cell types include L-selectin ligands, whereL-selectin ligands include sulfated forms of GlyCAM-1, CD34 andMAdCAM-1, and where L-selectin is displayed on the surface ofleukocytes; E-selectin ligands, where E-selectin ligandstetrasaccharides such as Sialyl-Lewis^(x) and Sialyl-Lewis^(a), andcutaneous lymphocyte-associated antigen, and where E-selectin is foundon the surface of endothelial cells; and antibodies to cell-specificcell surface molecules. Suitable attachment moieties include proteinsbound by cell surface integrins, where suitable attachment moietiesinclude laminin, collagen, fibronectin, tenascin, VCAM-1, MAdCAM-1,ICAM-1, ICAM-2, ICAM-3, fibrinogen, vitronectin, and the like. Suitableattachment moieties include integrins such as α4β1, α4β7, and the like.Suitable attachment moieties include antibodies specific formacromolecules displayed on a cell surface, where exemplary cell surfacemacromolecules include tumor-associated antigens; cell surfacereceptors; viral proteins (e.g., viral proteins displayed on the surfaceof a virus-infected cell); and the like.

In some embodiments, the attachment moiety is an antibody specific for atumor-associated antigen. Tumor-associated antigens (TAA) include, butare not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecularweight melanoma-associated antigen MAA, GD2, carcinoembryonic antigen(CEA), TAG-72, ovarian-associated antigens OV-TL3 and MOV18, TUAN,alpha-feto protein (AFP), OFP, CA-125, CA-50, CA-19-9, renaltumor-associated antigen G250, EGP-40 (also known as EpCAM), S100(malignant melanoma-associated antigen), p53, and p21ras.

Typically, the attachment surface comprises a plurality of attachmentmoieties, e.g., the attachment surface of a single micromirror comprisesfrom about 10 to about 10¹⁰ or more attachment moieties, e.g., fromabout 10 to about 100, from about 10² to about 10³, from about 10³ toabout 10⁴, from about 10⁴ to about 10⁵, from about 10⁵ to about 10⁶,from about 10⁶ to about 10⁷, from about 10⁷ to about 10⁸, from about 10⁸to about 10⁹, or from about 10⁹ to about 10¹⁰, or more, attachmentmoieties.

In some embodiments, the attachment surface comprises a plurality ofattachment moieties, wherein the plurality of attachment molecules ishomogeneous, e.g., all the attachment molecules on the surface of asingle mirror are identical. In other embodiments, the attachmentsurface comprises a plurality of attachment moieties, wherein theplurality of attachment moieties is heterogeneous, e.g., the pluralityof attachment moieties comprises at least two, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10, or more, different attachment moieties.

In some embodiments, the attachment moiety itself provides an externalstimulus to the cell, and the response of the cell to the attachmentmoiety is monitored by detecting cell membrane movement. In otherembodiments, the attachment surface comprises an attachment moiety; andan agent that provides an external stimulus to the cell.

In some embodiments, a single mirror is attached to a single cell. Inother embodiments, a plurality (two or more) of mirrors are attached toa single cell. An example of a situation in which use of multiplemirrors is useful is in analyzing cells in which different areas of thecell respond differentially to a given stimulus. Non-limiting examplesof such cells are polarized cells (e.g., columnar epithelial cellslining the gastrointestinal tract); and cells that have processes thatextend from the cell body (e.g., neurons, axons, dendritic cells, etc.).

In some embodiments, the invention provides arrays of micromirrors. Insome embodiments, each member micromirror in a subject micromirror arraycomprises an attachment surface with a different attachment moiety fromother array members. Mirrors can be purchased from commercial sources(see, e.g. Examples 1 and 2) and/or fabricated using any of a variety ofmethods known in the art. Suitable techniques include siliconmicromachining; SiNx micromachining; contact printing; dip penlithography; lift off techniques; and the like. In embodiments of theinvention, the micromirror comprises one of a wide variety of materialsknown to be suitable for such elements, for example a metallic materialsuch as nickel or a crystal material such as a silicon composition.

Sensors

Suitable sensors for use with embodiments of the invention include anydevice that is capable of detecting a reflected beam of light. A widevariety of such sensors are known in the art (see, e.g. U.S. Pat. Nos.5,233,197 and 7,176,459). Suitable sensors include, but are not limitedto, still cameras, video cameras, charge coupled devices (CCD) and thelike. The CCD camera is typically connected to an image analysiscomputer system for data storage and analysis. The scanning processestablishes a series of spatial mirror coordinates and mirror types ofall the mirrors on the sample. Typical sensors used in embodiments ofthe invention include those used in a wide variety of interferometricstudies and include for example those having the ability to observedfluorescently labelled materials (see, e.g. U.S. Pat. Nos. 7,365,858;6,381,025; 6,563,105; 7,088,458; and 7,298,496).

In some embodiments of the invention, two-dimensional arrays ofmicromirrors are used. Arrays of source beams of vertical cavity surfaceemitting laser (VCSEL) is directed at a two-dimensional array ofmirrors. VCSELs can provide a source beam having substantial lightintensity, without requiring additional lensing or amplification, in afocused area with low-beam divergence. The deflected beams areidentified and detected using a second array of micromirrors whichdefect the beams to an array of detectors (e.g., a two-dimensional arrayof photodetectors).

Computer Systems and Databases

Embodiments of the invention comprise databases of profiles of cellcharacteristics. Such databases will typically comprise profiles of cellmembrane movement of various cell types; cell membrane movement of cellsof various biological states; and cell membrane movement of cells inresponse to various biochemical events. A cell membrane characteristicprofile will contain, in addition to the cell membrane movement profile,one or more of the following cell parameters: cell type; biochemicalevent that stimulated the cell membrane movement; cellular environment,e.g., culture conditions, such as media composition and conditions,temperature, pH, osmolarity, as well as the physiological status of thecell. Thus, a typical cell characteristic profile can include the cellmembrane movement profile and at least one additional cell parameter. Inthis context, embodiments of the invention include the generation of areference library of such images and/or comparing a test image to one ormore images in a library of reference images.

The cell characteristic profiles and databases thereof may be providedin a variety of media to facilitate their use. “Media” refers to amanufacture that contains the expression profile information of thepresent invention. The databases of the present invention can berecorded on computer readable media, e.g. any medium that can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as compactdisc read only memory (CD-ROM); electrical storage media such as randomaccess memory (RAM) and ROM; and hybrids of these categories such asmagnetic/optical storage media. One of skill in the art can readilyappreciate how any of the presently known computer readable media can beused to create a manufacture comprising a recording of the presentdatabase information. “Recorded” refers to a process for storinginformation on computer readable medium, using any such methods as knownin the art. Any convenient data storage structure may be chosen, basedon the means used to access the stored information. A variety of dataprocessor programs and formats can be used for storage, e.g. wordprocessing text file, database format, etc.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage and processing means used to analyzethe information of the present invention. The minimum hardware of thecomputer-based systems of the present invention comprises a centralprocessing unit (CPU), input means, output means, and data storagemeans. A skilled artisan can readily appreciate that any one of thecurrently available computer-based systems are suitable for use in thepresent invention. The data storage means may comprise any manufacturecomprising a recording of the present information as described above, ora memory access means that can access such a manufacture.

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. One format for an output means ranks cellcharacteristic profiles possessing varying degrees of similarity to areference profile. Such presentation provides a skilled artisan with aranking of similarities and identifies the degree of similaritycontained in the test profile.

A subject database is useful for comparing a test profile to a referenceprofile that is stored in the database. Thus, the invention providesmethods of identifying or determining a characteristic of a cell,involving comparing a test profile of a cell with a database ofprofiles. For example, a cell profile that is generated in a clinicalsetting by analyzing a lung or other tissue biopsy sample (the “testprofile”) is compared to one or more reference profiles stored in thedatabase, where the reference profiles contain characteristics of normal(non-cancerous) lung cells, various types of cancerous lung cells, etc.Based on the comparison to the database, a determination of acharacteristic of the test profile is made.

The invention further provides methods of obtaining a cellcharacteristic profile, and methods of generating a database, orcollection, of cell characteristic profiles. The methods generallyinvolve detecting membrane movement of a cell, storing the membranemovement data on a computer readable medium (CRM), and linking the datawith at least one additional data about the cell (e.g., cell type,biological state, biological event which resulted in the membranemovement, cell medium conditions, and the like), thereby generating aprofile. The cell profile is recorded on a CRM. A database includes aplurality of such profiles. In some embodiments, the cell profile isrepresented in a visual format. In other embodiments, the cell profileis represented in a sound format. In other embodiments, the cell profileis represented in a graphical format.

Where the data are generated using a micromirror-based analytical systemas described above, the data are generated and stored in the form of thedegree of deflection of a reflected beam of light compared to a control.The data are obtained by measuring the cell membrane movement of a widevariety of cell types. The membrane movement of each cell type isrecorded under various conditions. As one non-limiting example, cellmembrane movement of a myocyte or other cell lineage is measured inmedia of various pH, in media containing various agents (e.g.,adrenaline, a calcium ionophore), in media containing various ionconcentrations, in media containing agents that induce a conditions thatmimics a disease state, under normal physiological conditions, and thelike. The cell characteristic is recorded for each condition, andinformation regarding the condition is entered into the database, suchthat the two pieces of information are linked. The information in thedatabase is searchable using terms for the cell type, and cellconditions. In another non-limiting example, cell membrane movement of aCD4⁺ T lymphocyte is recorded under normal physiological conditions(e.g., in serum), the cell membrane movement of a CD4⁺ T lymphocyte thatis infected with human immunodeficiency virus is recorded, and the cellmembrane movement of various T cell leukemias are recorded and stored inthe database.

Embodiments of the invention disclosed herein can be performed forexample, using one of the many computer systems known in the art. Forexample, embodiments of the invention include a database comprising aplurality of cell characteristic profiles recorded on a computerreadable medium, each of said cell characteristic profiles comprisingcell membrane movement data and at least one additional cell parameter.Additional cell parameters include, e.g., a cell type, a biologicalstate, a cell environment, and a stimulus. FIG. 17 illustrates anexemplary generalized computer system 202 that can be used to implementelements the present invention, including the user computer 102, servers112, 122, and 142 and the databases 114, 124, and 144. The computer 202typically comprises a general purpose hardware processor 204A and/or aspecial purpose hardware processor 204B (hereinafter alternativelycollectively referred to as processor 204) and a memory 206, such asrandom access memory (RAM). The computer 202 may be coupled to otherdevices, including input/output (I/O) devices such as a keyboard 214, amouse device 216 and a printer 228.

In one embodiment, the computer 202 operates by the general purposeprocessor 204A performing instructions defined by the computer program210 under control of an operating system 208. The computer program 210and/or the operating system 208 may be stored in the memory 206 and mayinterface with the user 132 and/or other devices to accept input andcommands and, based on such input and commands and the instructionsdefined by the computer program 210 and operating system 208 to provideoutput and results. Output/results may be presented on the display 222or provided to another device for presentation or further processing oraction. In one embodiment, the display 222 comprises a liquid crystaldisplay (LCD) having a plurality of separately addressable liquidcrystals. Each liquid crystal of the display 222 changes to an opaque ortranslucent state to form a part of the image on the display in responseto the data or information generated by the processor 204 from theapplication of the instructions of the computer program 210 and/oroperating system 208 to the input and commands. The image may beprovided through a graphical user interface (GUI) module 218A. Althoughthe GUI module 218A is depicted as a separate module, the instructionsperforming the GUI functions can be resident or distributed in theoperating system 208, the computer program 210, or implemented withspecial purpose memory and processors.

Some or all of the operations performed by the computer 202 according tothe computer program 110 instructions may be implemented in a specialpurpose processor 204B. In this embodiment, the some or all of thecomputer program 210 instructions may be implemented via firmwareinstructions stored in a read only memory (ROM), a programmable readonly memory (PROM) or flash memory in within the special purposeprocessor 204B or in memory 206. The special purpose processor 204B mayalso be hardwired through circuit design to perform some or all of theoperations to implement the present invention. Further, the specialpurpose processor 204B may be a hybrid processor, which includesdedicated circuitry for performing a subset of functions, and othercircuits for performing more general functions such as responding tocomputer program instructions. In one embodiment, the special purposeprocessor is an application specific integrated circuit (ASIC).

The computer 202 may also implement a compiler 212 which allows anapplication program 210 written in a programming language such as COBOL,C++, FORTRAN, or other language to be translated into processor 204readable code. After completion, the application or computer program 210accesses and manipulates data accepted from I/O devices and stored inthe memory 206 of the computer 202 using the relationships and logicthat was generated using the compiler 212. The computer 202 alsooptionally comprises an external communication device such as a modem,satellite link, Ethernet card, or other device for accepting input fromand providing output to other computers.

In one embodiment, instructions implementing the operating system 208,the computer program 210, and the compiler 212 are tangibly embodied ina computer-readable medium, e.g., data storage device 220, which couldinclude one or more fixed or removable data storage devices, such as azip drive, floppy disc drive 224, hard drive, CD-ROM drive, tape drive,etc. Further, the operating system 208 and the computer program 210 arecomprised of computer program instructions which, when accessed, readand executed by the computer 202, causes the computer 202 to perform thesteps necessary to implement and/or use the present invention or to loadthe program of instructions into a memory, thus creating a specialpurpose data structure causing the computer to operate as a speciallyprogrammed computer executing the method steps described herein.Computer program 210 and/or operating instructions may also be tangiblyembodied in memory 206 and/or data communications devices 230, therebymaking a computer program product or article of manufacture according tothe invention. As such, the terms “article of manufacture,” “programstorage device” and “computer program product” as used herein areintended to encompass a computer program accessible from any computerreadable device or media.

Of course, those skilled in the art will recognize that any combinationof the above components, or any number of different components,peripherals, and other devices, may be used with the computer 202.Although the term “user computer” is referred to herein, it isunderstood that a user computer 102 may include portable devices such asmedication infusion pumps, analyte sensing apparatuses, cellphones,notebook computers, pocket computers, or any other device with suitableprocessing, communication, and input/output capability.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Aspects of the invention disclosed herein can be found in Reed et al.,ACS NANO, 2(5): 841-846 (2008) and Reed et al., Nanotechnology 19:235101 (2008), the contents of which are incorporated by reference.

Example 1: High-Throughput Cell Nano-Mechanics with Mechanical ImagingInterferometry

This example provides an illustrative, high-throughput approach tomeasuring the nano-mechanical properties of a large number of cells inparallel, based on imaging interferometry in combination withreflective, magnetic probes attached to cells. In this example, wemeasure local elastic properties with applied forces of 20 pN to 20 nN,a spatial resolution of <20 nm, and a mechanical dynamic range ofseveral Pa up to ˜200 kPa. This disclosure provides evidence thatmechanical imaging interferometry (MII) is a sensitive and scalabletechnology for measuring the nanomechanical properties of large arrayslive cells in fluid. Illustrative data for NIH 3T3 and HEK 293Tfibroblasts as well as the effects of actin depolymerizing drugs aredisclosed.

This example demonstrates a nanomechanical probing method, calledmechanical imaging interferometry (MII), based on combining verticalscanning interferometry with reflective, magnetic probes attached tocells, that permits axially-oriented mechanical measurements of livecells with picoNewton force resolution over wide fields of view.Mechanical imaging interferometry (MII) has axial position repeatabilityof <20 nm over a very wide vertical range (millimeters), and can measurematerials with elastic moduli over the range of 50 Pa to 100+ kPa.Because the interferometric technique used is relatively insensitive tomagnification, we retain excellent positional resolution at fields ofview of up to or exceeding 740×570 microns, permitting simultaneousmeasurement of hundreds of probes. This allows throughput equal to orexceeding existing wide-field optical tracking techniques by severaltimes. Unlike these aforementioned methods, MII directly measures theposition of the bead on the cell membrane versus the substrate, and thusdetermine cell thickness very accurately, which is critical to accuratemechanical modeling of cells in some cases (see, e.g. Dimitriadis etal., (2002) Biophysical Journal 82, 2798-2810). Using softpolyacrylamide gels of known stiffness, we demonstrate an absolutemeasurement accuracy equal to that of AFM indentation in a similarexperimental configuration (6% standard error on a gel with Young'smodulus of 4 kPa, n=23)). Using MII we determine the quasi-staticmechanical properties of populations of NIH 3T3 and HEK 293Tfibroblasts, by probing large arrays of individual cells in parallel.The absolute values of the mechanical constants determined by MII are inexcellent agreement with results from a variety of other probingmethods.

The results show that MII is an effective, high throughput technique formeasuring cellular mechanical properties through indentation normal tothe cell surface. This represents a significant throughput advance overAFM, and other optical approaches, such as confocal microscopy ormicrofluidic optical stretchers (see, e.g. Guck et al., (2005)Biophysical Journal 88, 3689-3698), which cannot accurately measuremechanical properties of large arrays (hundreds) of cellssimultaneously, with single-cell specificity (see, e.g. Cheezum, M. K.,Walker, W. F. & Guilford, W. H. (2001) Biophysical Journal 81,2378-2388, Carter et al., (2005) Physical Biology 2, 60-72). Themechanical dynamic range and effective magnification of MII equals orexceeds existing wide-field optical particle tracking techniques (see,e.g. Fabry et al., (2001) Journal of Applied Physiology 91, 986-994),which implies that the two could be used in combination to conductrapid, fully-3D mechanical probing of large arrays of live cells.

Materials and Methods

Interferometer

The measurement of the microreflectors was performed on the Veecointerference microscope NT 1100 with a green diode (center frequency 535m) used for illumination and 20× 0.28NA Michelson through transmissivemedia (TTM) objective (see, e.g. Reed et al., (2006) PROCEEDINGS-SPIETHE INTERNATIONAL SOCIETY FOR OPTICAL ENGINEERING 0277-786X; 2006; VOL6293, p. 629301). The NT 1100 in principle is an optical microscope witha Michelson interference objective that allows for the observation ofnot only lateral features with typical optical resolution (1.16 μm forthe 20× objective) but also height dimensions below the scale of onenanometer (see, e.g. Olszak et al., (2001) Laser Focus World 37, 93-95).The Michelson interferometer is composed of a beam splitter, referencemirror and compensating fluid cell. The compensation cell is 0.7 mmthick bounded on both sides by 0.5 mm optical windows, thus matching theoptical path length of a reflected beam from the test chamber (i.e.matching the optical path difference between the arms). The CCD detectorarray is 640×480 pixels, which with a 20× objective produces a 315×240micron field of view and a spatial sampling of 500 nm. Measuredpositions of the reflectors with respect to the bottom of the samplechamber were corrected for the effect of dispersion in liquid, using agroup velocity at 535 nm wavelength and 30 nm bandpass of Ng=1.33 (see,e.g. Millard et al., (1990) Deep-Sea Research Part a-OceanographicResearch Papers 37, 1909-1926).

Cell Chamber

The cell chamber body was constructed from machined non-magneticstainless steel. Resistive heating elements with internal thermistors,driven by a feedback controlled power supply, were used to regulate thechamber temperature to within 0.5 degrees C. The fluid sample wascontained within a 13 mm diameter, 0.7 mm thick sub chamber, having a 1mm thick optical window on top and a 0.2 mm thick silicon floor. Fluidwithin the chamber could be exchanged through two peripheral infusionports, using a micro peristaltic pump capable of flow rates as low as 5μL min.

Microreflectors

Elemental nickel microspheres were obtained from Duke Scientific as adry powder with size distribution 2-10 μm in diameter. For eachexperiment approximately 0.1 mg of powder was mixed with 1 mL ofdistilled water. Smaller diameter particles were removed bysedimentation, resulting in a dilute suspension with size distribution˜5-10 μm. This suspension was diluted 4:1 with 0.2% poly-L-lysineaqueous solution (SIGMA) to inhibit aggregation and improve adhesion tothe cell bodies. The microreflector solution was shaken vigorouslybefore application to suspend any sedimented particles and reduceaggregates. 200 μL of the suspension was pipetted onto the sample (gelor cells) and the microreflectors allowed to settle for minute.

Magnetic Force Control

Magnetic force was applied to the microreflectors using a cylindricalrare earth magnet 7 mm in diameter by 21 mm long, oriented axially alongthe vertical direction below the test chamber. The magnet was positionedwith a feedback controlled motorized micrometer, capable of <10 μmaccuracy. The magnitude of magnetic flux perpendicular to the verticalaxis, as a function of axial distance, was measured with a miniatureHall probe. In the “off” position, the magnet was lowered to >4 cm belowthe sample, resulting in negligible field at the sample point. Themagnet was positioned coaxially with the optical path to ensure auniform magnetic flux across the viewing area (˜300 μm×300 μm with the20× objective). The force applied to the nickel microreflectors as afunction of magnet position was determined using microcantilever arraystipped with elemental nickel or several uniformly magnetic microspheres(Compel 8 um carboxylated microspheres, BANGS LABS). Eachmicrocantilever is 500 microns long by 100 microns wide and 0.9 micronsthick, with a nominal spring constant of 0.01 N/m. These commerciallyavailable arrays were produced by the IBM ZURICH RESEARCH LABORATORIESusing a proprietary dry etch, silicon-on-insulator (SOI) process. Usingthe optical profiler, the deflection of the reference cantilever couldbe determined to better that 1 nm. The volume magnetic moment for purenickel (55 emu/g) was assumed for both the microreflectors and thenickel film deposited on the cantilever tips. Pure nickel is completelymagnetically polarized at field strengths of 200 G and higher, while thelowest field strength used in measurements was 500 G. Precedingmeasurements, the magnet was raised to with 1.5 mm of the sample,corresponding to a ˜2 kG flux at the sample point, to ensure that themicroreflectors' magnetic moments were oriented axially.

Polyacrylamide Gel Tests

5% acylamide/0.15% bis-acrylamide and 5% acrylamide/0.05% bis-acrylamidegels were cast between a microscope slide and cover slip, using 40 μmtape as a spacer, using standard conditions (see, e.g. Engler et al.,(2004) Surface Science 570, 142-154). A 5×5 mm section of each gel wasremoved with a scalpel and placed inside the fluid test chamber formeasurement. Sample in the test chamber were allowed to equilibrateovernight in 1×TBE buffer, pH 7.5, the same buffer used to prepare thegels. All measurements were conducted under buffer.

Cell Viscoelastic Measurements

The population measurements of the NIH 3T3 fibroblasts and the HEK 293Tfibroblasts were conducted over several consecutive days. Both celltypes were cultured at the same time on a series of poly-L-lysine coated0.20×10 mm round glass coverslips, in 1×DMEM with 10% fetal bovine serumin a laboratory incubator under standard cell culture conditions.Preceding measurement, a single round coverslip containing cells wouldbe removed from the culture dish and quickly placed in the microscopetest chamber. Microreflectors would be added to media from the originalculture dish, which would then be pipetted slowly onto the cells and thesample chamber sealed. Cells were allowed to equilibrate in the testchamber for 30 minutes before measurement. The average radius of themeasured microreflectors was 3.79 μm, and the average applied force was190 pN. The average cell height was 8.1 μm and the average maximumindentation depth per cell was 660 nm. The following factors showed nosignificant difference between the two populations on the basis of ANOVAanalysis: Cell height, applied force, reflector radius and maximumindentation depth/radius.

The three constants for a viscoelastic solid were determined for eachmeasurement by fitting the time-dependent force-displacement curves ofthe microreflectors to the following equation (see, e.g. Cheng et al.,(2005) Mechanics of Materials 37, 213-226):

${\delta (t)} = {\frac{3}{4}\sqrt[3]{3{F^{2}/4}{RE}_{1}^{2}}\left\{ {\left( {{- \frac{E_{1}}{E_{2}}}e^{- {({E_{2}{t/3}\eta})}}} \right) + \left( \frac{E_{1} + E_{2}}{E_{2}} \right)} \right\}^{2/3}}$

Our measurements assumed Poisson's ratio for the cell, v=0.5. Curveswere fitted using the Levenberg-Marquardt non-linear least squaresprocedure (Origin Labs). The z-statistic was used to compare thelog-transformed sample means and determine p values. The Bartlett testwas used to confirm homogeneity of the compared sample variances.

To remain within the semi-infinite layer assumptions of this model, wepresent fits only for observations where the minimum cell thickness atthe location of the microreflector was 3 μm or greater. A majority ofthe measurements fit the model elastic constants E₁ and E₂ well, havinga relative standard error of fit <25%. There was larger uncertainty inthe viscosity factor, η, with only 30% of the fits having a relativestandard error <25%. The error in η is mainly due to the temporalsampling rate of 0.1 Hz, which did not adequately resolve the rapidindentation of the microreflectors on the softest cells.

Cytochalasin B Measurements

Cells were prepared for measurement as described above. A continuousinfusion of fresh media into the sample chamber, warmed to 37 C andpre-saturated with 5% CO₂, was maintained at all times. The rate ofinfusion was 5 μL/min, equivalent to exchanging the entire volume of thetest chamber in 20 minutes. Cells were equilibrated under flow for 45minutes before measurement. Flow was halted during each measurementcycle, which lasted approximately 200 seconds. The first, a media-onlymeasurement was conducted at 1 hour, immediately followed byintroduction of cytochalasin B into the infusing media. Initially, thecytochalasin B was dissolved in DMSO and diluted in DMEM to produce astock concentration 1000× the working concentration (1 μM or 10 μM). Atthe appropriate time, the stock solution was introduced into theinfusion media reservoir to produce the desired final concentration.

Mechanical Dynamic Range and Throughput.

The range of force achievable on an 8 micron diameter nickelmicroreflector was approximately 20 pN up to 20 nN. We could obtainaxial measurement precision of the microreflector versus the samplesubstrate of <20 nm, which is sufficient to compute the elastic modulusof soft materials with less than 5% error, using the linear Hertz modelfor a spherical indenter (see, e.g. Lim et al., (2006) Journal ofBiomechanics 39, 195-216). For the purpose of this analysis, it wasassumed that the bead diameter and magnetic force can be determined toarbitrary precision.

In both MII and standard wide field bead tracking experiments, for agiven bead diameter and measurement time, throughput is limited by thenumber of probes in the field of view. We were able to obtainmeasurement precision of <20 nm using a 20× 0.28NA objective and a 0.5×demagnifier, with a CCD detector array having 640×480 pixels, whichproduced a 600×440 micron field of view and a spatial sampling of 952nm. In comparison, wide-field optical magnetic twisting cytometry (OMTC)uses a 10×0.2NA objective and a camera with 780×600 pixels, whichproduces a 450×350 micron field of view and a spatial sampling of 570 nmper pixel. Therefore, if we used a CCD with 780×600 pixels and the same952 nm spatial sampling, we would obtain a field of view of 742×571microns, which equates to roughly 3× the measurement area vs. OMTC.

Results and Discussion

Mechanical imaging interferometry (MII) makes use of sphericalmicroreflectors fixed to the cell membrane, that act as nanoscopicdisplacement probes. We developed a special liquid force cell for thesemeasurements using a Michelson interferometer (FIG. 1A). Measuring livecells in culture required the placement of a liquid-filled compensationcell in the reference arm of the interferometer. Dimensions of thecompensation cell were adjusted to exactly match the optical path lengthbetween the test and reference arms. Cells are evaluated in a sealedenvironmental chamber maintained at 5% CO₂, 37° C., with infusion portsfor exchanging media and the introduction of drugs and other chemicals.The microreflectors were pure nickel, ranging from 6 to 10 microns indiameter.

During each measurement, the objective head is scanned vertically fromthe surface to a height 40 microns above the surface, such that eachpoint in the volume passes through focus. The interferometer is alignedso that the interference intensity distribution along the verticalscanning direction has its peak (best fringe contrast) at approximatelythe best focus position. The vertical-axis position of eachmicroreflector is determined as the location of the coherence peakwithin the scan. By measuring microreflectors of known height fixed to asolid substrate in liquid, we determined that the z-axis measurementrepeatability was <20 nm. Magnetic forces applied to the nickelmicrospheres were calibrated with a ferromagnetic-tipped microcantileverarray, having a known spring constant (k=0.01 N/m) and magnetic moment.The range of force achievable on an 8 micron diameter nickelmicroreflector was approximately 20 pN up to 20 nN. A criticalconsideration for cell nanomechanical measurements is the dynamic rangeof the measurement technique. Mammalian cells exhibit a wide range ofYoung's moduli, from as soft as 10 Pa to as stiff as 100 kPa (see, e.g.Balland et al., (2006) Physical Review E 74). We estimate that MII caneffectively measure samples with elastic moduli that vary from severalPa up ˜200 kPa, as currently configured.

To validate our approach, we first tested the microreflectors on 40micron thick, soft polyacrylamide (PA) gels under liquid, whichsimulated the cell body (FIGS. 2A-2D). We recorded the verticaldisplacement of the microspheres in response to a series of increasingforces. The resulting force-displacement curves fit the Hertz contactmodel (see, e.g. (Lim et al., (2006) Journal of Biomechanics 39,195-216) for a spherical indenter well. For the 40 micron-thickpolyacrylamide gels, the measured values for Young's modulus werelinearly proportional to the crosslinker concentration, as expected, andthe range of absolute values and measurement precision (1,530 S.E.+/−128Pa (n=22) and 4,020 S.E.+/−270 Pa (n=23)) agree well with similarmeasurements by others using AFM and bulk techniques (see, e.g. Mahaffyet al., (2004) Biophysical Journal 86, 1777-1793; Mahaffy et al., (2000)Physical Review Letters 85, 880-883; Engler et al., (2004) BiophysicalJournal 86, 617-6).

Next, we measured the mechanical properties of live mouse NIH3T3 andhuman HEK293T fibroblasts. In the perfusion chamber, the microreflectorson top of individual cells or cell layers appeared as distinct objectsin the interferometer image (FIGS. 3A and 3B). Similar to the PA geltests, we applied a series of step forces and recorded verticalreflector displacements. The indentation and recoil was proportional tothe applied force, as expected, but the cell bodies showed a distinctviscoelastic response, versus the purely elastic behavior of the PAgels. This is most clearly seen as ‘creep behavior’ of themicroreflectors in response to a step change in force (FIG. 4).

Live cells are known to exhibit complex frequency-dependent viscoelasticproperties (see, e.g. Trepat et al., (2007) Nature 447, 592-U7;Lenormand et al., (2004) Journal of the Royal Society Interface 1,91-97; Desprat et al., (2005) Biophysical Journal 88, 2224-223328). Forthe purpose of this study were primarily interested in determining theequilibrium elastic modulus of the cells. To this end, we used a3-factor linear viscoelastic solid model to parameterize the cell'sresponse (see, e.g. Cheng et al., (2005) Mechanics of Materials 37,213-226). This type of model has been used to characterize theviscoelastic properties of cells and soft polymers (see, e.g. Lim etal., (2006) Journal of Biomechanics 39, 195-216). It consists of twosprings and a dashpot, specified by two elastic constants (E1, E2) andone viscous constant (η). The stress-bearing capacity of thecytoskeleton is represented by a short term-response, E1, and a slowerresponse, E2. The response of the spring element E₂ is delayed byviscous drag. Our ability to resolve the viscous constant is limited bythe 0.1 Hz temporal sampling rate, but this does not impact our abilityto measure the equilibrium elasticity, which is given by E₁ and E₂. Afull temporal characterization of the cellular viscoelastic response isbeyond the scope of this study, however, as we discuss below, MII isfully compatible with the cyclic probing methods commonly used todetermine frequency-dependent mechanical moduli. For both cell types,the population distributions of the viscoelastic constants werelog-normally distributed, with geometric standard deviations of ˜1.5logs. This result is in agreement with recent reports (see, e.g. Fabryet al., (2001) Journal of Applied Physiology 91, 986-994; Balland etal., (2006) Physical Review E 74; Maksym et al., (1999) American Journalof Respiratory and Critical Care Medicine 159, A470-A470). The means andstandard errors of the log-transformed pooled measurements for both celltypes are shown in FIG. 5. The mechanical constants for the HEK293Tcells are consistently lower than for the NIH3T3 cells, although thedifference was statistically significant at the >95% level only for thedelayed elastic constant E2.

We evaluated the behavior of NIH3T3 cells (n=30) exposed to a low dose(1 μM) of cytochalasin B, which inhibits actin polymerization. At lowdoses (0.1-1 μM), cytochalasin B does not produce large changes in themorphology of fibroblasts, although it does inhibit cell migration (see,e.g. Rotsch et al., (2000) Biophysical Journal 78, 520-535; Yahara etal., (1982) Journal of Cell Biology 92, 69-78), and AFM indentationstudies have reported minimal, if any, measurable change in Young'smodulus (see, e.g. Rotsch et al., (2000) Biophysical Journal 78,520-535). In contrast, we determined that treated cells were lesselastic. For the population, the mean of the log-transformeddistribution of E1 was 3.19 before treatment and 2.92 after (p=0.10).Similarly, the means of E2 were 2.30 before and 2.02 after (p=0.04), andthe means of the viscous constants were 3.39 before and 3.36 after(p=0.47) (FIG. 6A). While the population difference was statisticallysignificant only for E2, on a matched, individual cell basis, theelasticity was consistently lower after treatment (E1 10/13 lower, E27/10 lower), while viscosity was not (η 6/11 lower). Qualitatively, 1 μMcytochalasin produced only a slight change in cell morphology up to 45minutes, as expected, while the indentation profiles show a clear changein some cells and not in others (cells 2 and 3 in FIG. 6B).

Measurements of live fibroblast cells provide a direct comparison of MIIto AFM and other nano-indentation methods. The magnitude of the elasticconstants and the response to cytochalasin B determined by MII were inexcellent agreement with reported results for fibroblasts, determined byAFM, bead-tracking microrheology and microplate traction (see, e.g. Limet al., (2006) Journal of Biomechanics 39, 195-216). The temporalsampling rate in our experiments was 0.1 Hz, effectively limiting ourmechanical measurements to quasi-static. Therefore, while the averageviscous constant we determined for both fibroblast cell types is similarto that reported by other methods (see, e.g. Lim et al., (2006) Journalof Biomechanics 39, 195-216; Koay, E. J., Shieh, A. C. & Athanasiou, K.A. (2003) Journal of Biomechanical Engineering-Transactions of the Asme125, 334-341), we cannot effectively determine η for cells with a verylow viscosity (<˜1 kPa s). This resulted in the relatively larger errorin fitting the viscous constant versus the elastic constants in ourthree factor mechanical model.

On the other hand, MII is very well suited to investigate longertimescale mechanical responses, including “active” behavior such ascytoskeletal remodeling and cell motility, because unlike most competingtechnologies, it does not require the filtering out of low frequencymotions to achieve accuracy, and the absolute height of the probe overthe substrate is measured with high precision (<0.2%) every measurementcycle. In fact, a subset of the creep curves we recorded showedtime-varying behavior consistent with an active mechanical response,such as the cell lifting the microreflector several hundred nanometerswhile under load. The duration of the force cycle, 100 s, was within thetime-scale of active mechanical responses by the cell, such aslamellepodial extension/retraction, and cytoskeletal tensioning bymolecular motors (see, e.g. Tamada et al., (2004) Developmental Cell 7,709-718; Giannone et al., (2004) Cell 116, 431-443).

Optical magnetic twisting cytometry (OMTC) developed by Fredberg andcolleagues (see, e.g. Smith et al., (2003) American Journal ofPhysiology-Lung Cellular and Molecular Physiology 285, L456-L463; Fabryet al., (2001) Journal of Applied Physiology 91, 986-994; Mijailovich etal., (2002) Journal of Applied Physiology 93, 1429-1436; Maksym et al.,(2000) Journal of Applied Physiology 89, 1619-1632) is the only othertechnique capable of measuring the mechanical properties of individuallive cells with a throughput and sensitivity comparable to MII. We donot include in this discussion the OMTC-related studies that utilizevery high optical magnifications (see, e.g. Hu, S. H., Eberhard, L.,Chen, J. X., Love, J. C., Butler, J. P., Fredberg, J. J., Whitesides, G.M. & Wang, N. (2004) American Journal of Physiology-Cell Physiology 287,C1184-C1191).

The primary difference between the two methods is that with MII themechanical properties of the cell are measured by indentationperpendicular to the cell surface, whereas OMTC measures mechanicalshear in the x-y plane of the cell membrane. Aside from theorientation-specific mechanical information obtained, z-axis indentationvs. surface twisting (shearing) has several advantages. It does notrequire that the probe be tethered to cell surface receptors as in thecase of twisting measurements (see, e.g. Mijailovich et al., (2002)Journal of Applied Physiology 93, 1429-1436), and because themagnetizing force and the indenting force are aligned in MII, there isno limit to the maximum magnetic field which can be applied. In OMTC theactuating (twisting) field is perpendicular to the probe's magneticmoment, and thus is limited to less than ˜100 Gauss in order to avoiddemagnetizing the probe. In both methods, the measurement of absolutemechanical constants using surface-bound probes requires assumptionsabout the probe-cell contact area which are difficult to validate insitu; this is a concern common to magnetic/optical bead twisting andpulling methods, as well as AFM in some cases.

We estimate that both methods have a similar dynamic range of measurableelastic moduli, from tens of Pascals to 100+ kiloPascals. The effectivethroughput, which is limited by the field of view, is similar andperhaps several times larger for MII vs. OMTC, we believe. OMTC claimspositioning accuracy of 5-10 nm versus 10-20 nm for VEII, although inOMTC this is achieved using phase-locked detection, not a requirementbut easily implemented in MII. Unlike OMTC, AFM, and some other opticaltechniques, MII determines absolute cell height every measurement, to˜0.3% accuracy, and has a vertical range of millimeters. This allows MIIto capture dynamic changes in cell shape and multi-cell structures,without compromising sensitivity. Also, knowing the thickness of thecell below the probe is critical to accurate mechanical modeling ofcells in some cases (see, e.g. Dimitriadis et al., (2002) BiophysicalJournal 82, 2798-2810).

OMTC utilizing phase-locked detection has a wide temporal dynamic range,from 0.01 Hz to 100+Hz. Our study utilized a temporal sampling rate of0.1 Hz. However, this is not a fundamental limitation of MII. We haveused phase-locked detection to measure nanometer motion of MEMSstructures up to 1 MHz with the MII optical system (see, e.g. Reed etal., (2006) Nanotechnology 17, 3873-3879), and we expect that this couldeasily be translated to live cell measurements.

Finally, MII may prove to be more scaleable than OMTC, which has beenoptimized over several years to achieve spatial sensitivity of 5-10 nm,or 0.008 fractional pixels, which studies suggest is at or near thepractical limit of non-interferometric particle tracking (see, e.g.Cheezum, M. K., Walker, W. F. & Guilford, W. H. (2001) BiophysicalJournal 81, 2378-2388; Carter et al., (2005) Physical Biology 2, 60-72).On the other hand, the accuracy of measurement in the z-axis usingvertical scanning interferometry is theoretically insensitive tomagnification, for surfaces with low curvature (see, e.g. Olszak et al.,(2001) Laser Focus World 37, 93-95; Olszak et al., (2003) OpticalEngineering 42, 54-59). At its limit, interferometric microscopy canoperate with magnifications as low as 1×, or approximately 5 mm×5 mmfield of view. If this level of resolution could be achieved withmicroreflectors, spherically-shaped or otherwise, it would translate tothe ability to perform cell mechanical measurements simultaneously overan area in excess of 600× conventional methods. This would enablelongitudinal time studies of mechanical properties, where not onlysingle-cell, but simultaneous cell-cell interaction and the effect oflong-scale (hundreds of microns) physical or chemical gradients can beobserved.

The primary conclusions of this study are as follows: MII is capable ofaxially tracking magnetic microreflectors, with <20 nm spatialprecision, in the optically complex environment of live cell culture.Combined with a 20 pN-to-20 nN range of achievable forces on a typicalmicroreflector, MII can determine the elastic moduli of live cells,through nano-indentation, over a wide dynamic range, from several Pa upto ˜200 kPa. MII attained excellent positional resolution at loweffective magnification (spatial sampling ˜500-900 nm per pixel),permitting simultaneous measurement of up to 100 probes.

Using soft polyacrylamide gels of known stiffness, we demonstrate anabsolute measurement accuracy slightly exceeding that of AFM indentationin a similar experimental configuration (6% standard error on a gel withYoung's modulus of 4 kPa, n=23). Using MII we determine the quasi-staticmechanical properties of populations of NIH 3T3 and HEK 293Tfibroblasts, we determined the absolute values of the mechanicalconstants which are in excellent agreement with results from othernanomechanical probing methods.

Our results show that MII is an effective, high throughput technique formeasuring cellular mechanical properties through indentation normal tothe cell surface. This represents a significant throughput advance overAFM, and other optical approaches, such as confocal microscopy ormicrofluidic optical stretchers, which cannot accurately measuremechanical properties of large arrays (hundreds) of cellssimultaneously, with single-cell specificity (see, e.g. Cheezum, M. K.,Walker, W. F. & Guilford, W. H. (2001) Biophysical Journal 81,2378-2388; Carter et al., (2005) Physical Biology 2, 60-72). Themechanical dynamic range and effective magnification of MII equals orexceeds existing wide-field optical particle tracking techniques (see,e.g. Fabry et al., (2001) Journal of Applied Physiology 91, 986-994),which implies that the two could be used in combination to conductrapid, fully-3D mechanical probing of large arrays of live cells.

Example 2: Live Cell Interferometry Reveals Cellular Dynamism DuringForce Propagation

This example provides an illustration of how to employlive-cell-interferometry (LCI) to visualize the rapid response of awhole cell to mechanical stimulation, on a time scale of seconds, and wedetect cytoskeletal remodeling behavior within 200 seconds. Thisbehavior involved small, rapid changes in cell content and minisculechanges in shape; it would be difficult to detect with conventional orphase contrast microscopy alone, and is beyond the dynamic capability ofAFM. We demonstrate that LCI provides a rapid, quantitativereconstruction of the cell body with no labeling, which is highlycomplementary to traditional microscopy and flow cytometry, whichrequire cell surface marker detection and/or destructive cell fixationfor labeling.

Briefly, mammalian cells exhibit continuous regional motion and shapechanges controlled by a dynamic cytoskeleton. The movements of a cellare orchestrated by a dynamic cytoskeleton that extends from the fluidlipid bilayer and underlying actin cortex to deep within a cell. Themechanical scaffold of each cell is composed of relatively stiffcomponents including actin microfilaments, intermediate filaments,microtubules and a myriad of crosslinking, motor and regulatory proteinsthat maintain structure and control dynamism. Numerous studies linkthese cytoskeletal structures to biochemical signal transductionpathways to regulate cellular processes including adhesion, motility,gene expression and differentiation (see, e.g. Kumar et al., BiophysicalJournal 2006, 90, 3762-3773; Matthews et al., Journal of Cell Science2006, 119, 508-518; Felsenfeld et al., Nature Cell Biology 1999, 1,200-206; Lee et al., Nature 1999, 400, 382-386; Yauch et al., Journal ofExperimental Medicine 1997, 186, 1347-1355; Felsenfeld et al., Nature1996, 383, 438-440; Chicurel et al., Nature 1998, 392, 730-733). A keychallenge that remains is connecting specific structures and signalingto the changing biophysical properties of the cell and vice-versa. Acritical issue in probe-based measurements of cell mechanics, such asAFM and optical/magnetic tweezers, is the speed and degree to which amechanical force exerted by a probe propagates across the cell body(see, e.g. Van Vliet et al., ACTA MATERIALIA 2003, 51, 5881-5905). Thisis seldom documented because observing deformation of the entire cellbody with required speed and accuracy (˜1% local deformation) isexperimentally complex, for a variety of reasons. For example, in mostcases AFM cannot accurately image the membrane of an entire mammaliancell at the rate of 1-2 Hz, due to the softness of the cell membrane.Bead-based approaches, such as magnetic and optical traps, track themotion of the bead itself and not the surrounding cellular materialwhich is unlabeled. Labels can be introduced but this adds considerablecomplexity. With phase contrast methods organelles themselves can serveas displacement probes, but they are not uniformly distributed, which isa major constraint when investigating dynamic structure is the cellperiphery such a filopodia.

These problems are addressed by the imaging technique disclosed herein,called live cell interferometry (LCI), to directly assess thepropagation of strain throughout a single cell in response to locallyapplied force. By measuring changes in optical path length distributionacross many points within the cell simultaneously, we could determinethe corresponding redistribution of cellular constituents, and thuscould quantify responses of the cell body distal to the point of appliedforce in real time and without labeling. The imaging system (FIG. 1A)consists of an optical microscope with a Michelson interferenceobjective, a fluid-filled live-cell observation chamber with areflective floor, and a matched reference chamber containing only fluid.It operates as follows: The illumination wavefront incident on theobservation chamber travels through the transparent fluid (culturemedia) and the transparent cell body, and is returned to theinterferometer by the reflective substrate. The index of refraction ofthe culture media and the cell body are so close in value that there isvery little reflection of the light from the cell-fluid interface.However, the difference in the index of refraction of the fluid and cellis sufficiently large for the detection of changes in optical pathlength introduced by the cell as the light travels through it and isreflected back from the substrate. The optical path length is thegeometrical path the wavefront travels times the index of refractiondistribution n(z) of the media at a given point. Because the lightreflects from the substrate and returns to the objective, it travelstwice the same geometric distance z, the optical path length can beexpressed as:

Sample Optical Path Length=2∫zn(z)dz  (1)

While for the light traveling through the reference chamber fluid,having a uniform distribution of index of refraction, the optical pathlength it is simply:

Reference Optical Path Length=2zn _(fluid)  (2)

In the LCI technique, this difference in optical path is recorded as ashift in phase between the sample observation and reference beams (see,e.g. Creath K; Schmit J. Phase shifting interferometry. In: Guenther B,editor. Encyclopedia of Modern Optics. Boston: Elsevier Academic Press;2005. p. 364-374). The resulting LCI phase image shows a distribution ofthe optical path lengths in the field of view of the objective. Obtainedin this way the signature of the cell shows that the optical path lengthis longer through the cell versus fluid by about 0-400 nm (FIG. 7). Thisdata agrees with the assumption that the index of refraction of thefluid is about 1.33, the index of refraction for the cell body is1.4-1.5 and the maximum thickness of the cell is about 5-8 microns.Thus, the measured optical path length represents the distribution ofcells thickness and material index of refraction together.

By comparing optical path length images taken at two consecutive timepoints, we determined very precisely local shifts of material within thecell. This is illustrated in FIG. 7. We were able to reliably detectchanges in optical path length as small as ˜1 nanometer. Since the cellbody appears to be between 0 to 400 nm in optical thickness, thiscorresponds to the ability to detect <1% changes in optical path lengthover large portions of the cell.

We recorded shifts in optical thickness in regions adjacent to magneticmicrospheres undergoing cyclical indentations at 0.05 Hz for 200 s or 10cycles (FIGS. 8A-8F). Two 5 μm diameter microspheres were evaluatedsimultaneously on an elongated NIH 3T3 fibroblast. The maximum appliedforce was ˜200 pN for each microsphere. The mechanical linkage betweenthe force-driven and undriven regions of the cell was measured as thechange in the optical thickness profiles over each indentation cycle. Ashift in cell content was not readily apparent in either the intensityimage or the LCI image itself, but was detected by comparing thedifference between two LCI images. This differential LCI image provideda quantitative measure of the redistribution of material in the cell inresponse to the indenting body for any two time points. In theseexperiments two features became apparent: first, the strain field due tothe indenting sphere extends across the entire cell, in a pattern thatsuggests displacement of core underlying, rigid structures (FIG. 9); andsecond, the indentation produces an immediate, synchronized andlaterally continuous increase in material at the cell periphery,consistent with pressure-driven flow. Detecting these rapidrearrangements in the local material density would be difficult withnon-interference-based optical methods, and is beyond the dynamiccapability of AFM. Optical waveguides have been used to observe thenanometer-scale deformation of a metallic substrate surface caused by aplant fungus. This method is confined to reflective surfaces however,whereas LCI measures subtle index of refraction changes of a volume oftransparent material (the cell body).

We analyzed the time-dependence of the content shift between specificregions of the cell by measuring the change in average optical thicknesswithin four sub-regions of the cell body (FIG. 8A left). The undrivenregions responded at the same frequency as the driven regions, but witha temporal delay, as would be expected from a viscoelastic material(FIG. 8A). The amplitudes of motion of both the driven and undrivenregions increased with time (FIGS. 10A and 10B). In the driven region A1the amplitude peaked at ˜100 sec and leveled off, while the amplitude ofthe other region, B1, peaked later (˜150 sec), and then began todecline. This behavior suggests that the local compliance of the cellchanged with time, and that this change was distributed heterogeneouslywithin the cell.

After 200 s of cyclically applied force, there was a noticeable,non-transient redistribution of material within the cell body. Themagnitude of this shift was larger than the transient response, withmaximum local changes in content of 10-15%, but was still observableonly in the differential LCI images (FIG. 11). Material accumulated inthe center of the cell, preferentially along the “backbone” of the longaxis. Lamellepodia also formed at either end of the cell parallel to thelong axis. There was an opposing loss of material at the edges of thecell adjacent to the two microspheres, parallel with the short axis ofthe cell. Notably, the regions with the largest decrease in materialover 200 s corresponded to the regions of the cell which saw the mosttransient change in content during each earlier force cycle. The forcedmotion was ended at t=200 s and the cell was imaged again at t=400 s. Bythis time, the pattern of material accumulation within the cell hadlargely reversed, the lamellepodia parallel to the long axis retractedand material accumulated adjacent to each microsphere, parallel to theshort axis of the cell. This behavior suggests an active remodelling ofthe cytoskeleton in response to cyclic loading (see, e.g. Matthews etal., Journal of Cell Science 2006, 119, 508-518). Local force-inducedremodeling is known to begin within tens of seconds following amechanical stimulus (see, e.g. Hayakawa et al., Experimental CellResearch 2001, 268, 104-114). The edges of the cell retracted and thenre-extended at a linear velocity of approximately 180 nm per second,which is consistent with active, actinomyosin-driven motion. Theaccumulation of material along the cell ‘backbone’ is also consistentwith enhanced cytoskeletal contractility, known to occur in some cellsfollowing forced stretching or similar mechanical deformations (see,e.g. Hayakawa et al., Experimental Cell Research 2001, 268, 104-114;Deng et al., American Journal of Physiology-Cell Physiology 2004, 287,C440-C448; Smith et al., American Journal of Physiology-Lung Cellularand Molecular Physiology 2003, 285, L456-L463). There was a considerabletransient change in content near the cell periphery during each forcecycle, the same areas that showed the greatest non-transient shift inmaterial accumulation. Overall, this behavior demonstrates a globalcoordination and reorientation of the cell structure in response to alocal, cyclically-applied stress. The mechanisms triggering this contentrearrangement could include both phosphatase-integrin complex activation(see, e.g. Matthews et al., Journal of Cell Science 2006, 119, 508-518;Felsenfeld et al., Nature Cell Biology 1999, 1, 200-206; Yauch et al.,Journal of Experimental Medicine 1997, 186, 1347-1355) and membranestretch-activated ion channel function (see, e.g. Matthews et al.,Journal of Cell Science 2006, 119, 508-518; Felsenfeld et al., NatureCell Biology 1999, 1, 200-206; Yauch et al., Journal of ExperimentalMedicine 1997, 186, 1347-1355). The latter have been shown induce tailretraction in migrating keratocytes, and are known to trigger acalcium-dependant signaling cascade that results in the phosphorylationof myosin, leading to retraction of filipodia (see, e.g. Matthews etal., Journal of Cell Science 2006, 119, 508-518; Felsenfeld et al.,Nature Cell Biology 1999, 1, 200-206; Yauch et al., Journal ofExperimental Medicine 1997, 186, 1347-1355). The synchronized andlaterally continuous increase in material at the periphery of the cellin response to indentation seems likely to have disturbed the cellmembrane in these areas, and suggests that membrane stretch mechanismsneed to be considered (see, e.g. Matthews et al., Journal of CellScience 2006, 119, 508-518; Sheetz et al., Annual Review of Biophysicsand Biomolecular Structure 2006, 35, 417-434).

In many magnetic/optical tweezer experiments, and some AFM experiments,the probe is coated with a peptide ligand to promote specific attachmentto cell surface receptors. In theory, this allows a direct mechanicallink to the cytoskeleton, which could propagate an applied force toother regions of the cell and a vehicle for studying whole cellmechanical dynamics. However, in practice the degree of attachment tothe cytoskeleton is seldom confirmed, and conflicting results have beenreported regarding the degree of force propagation (see, e.g. Matthewset al., Journal of Cell Science 2006, 119, 508-518; Bausch et al.,Biophysical Journal 1999, 76, 573-579). In some cases experimenters haveobserved the displacement of organelles or injected particles to inferthe strain field, though this produces only a sampling and not a globalmeasurement (see, e.g. Hu et al., American Journal of Physiology-CellPhysiology 2003, 285, C1082-C1090; Bausch et al., Biophysical Journal1998, 75, 2038-2049). Various labeling schemes can make this sort ofmeasurement more comprehensive, but often require engineered cell linesor tedious labeling procedures that limit their use. LCI is compatiblewith these existing approaches, and addresses one of their significantweaknesses by globally capturing the redistribution of cell material inresponse to force. This spatially- and temporally-detailed tracking ofcell material also may have utility in improving computational models ofcell mechanics, which are now largely phenomenological (see, e.g. Lim etal., Journal of Biomechanics 2006, 39, 195-216; Stamenovic et al.,Journal of Theoretical Biology 1996, 181, 125-136; Wang et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 2001, 98, 7765-7770; Canadas et al., Journal of BiomechanicalEngineering-Transactions of the Asme 2006, 128, 487-495), and havedifficulty assigning specific model parameters to discrete mechanicalstructures within the cell.

Exameplary Methods and Materials

Interferometer.

The measurement of the microspheres was performed on the Veecointerference microscope NT 1100 with a green diode (center wavelength535 m) used for illumination and 20× 0.28NA Michelson throughtransmissive media (TTM) interference objective (see, e.g. Reed et al.,PROCEEDINGS-SPIE THE INTERNATIONAL SOCIETY FOR OPTICAL ENGINEERING0277-786X; 2006; VOL 6293 2006, 6293, p. 629301). The NT 1100 inprinciple is an optical microscope with a Michelson interferenceobjective that allows for the observation of not only lateral featureswith typical optical resolution (1.16 for the 20× objective) but alsoheight dimensions below the scale of one nanometer (see, e.g. Olszak etal., Laser Focus World 2001,37, 93-95). The Michelson interferometer iscomposed of a beam splitter, reference mirror and compensating fluidcell. The compensation cell is 0.7 mm thick bounded on both sides by 0.5mm optical windows, thus matching the optical path length of a reflectedbeam from the test chamber (i.e. matching the optical path differencebetween the arms). The CCD detector array is 640×480 pixels, which witha 20× objective produces a 315×240 micron field of view and a spatialsampling of 500 nm. The phase shifting interferometry (PSI) method wasused to capture phase images of the cell bodies in situ. Duringmeasurement, a piezoelectric translator decreases the light path a smallamount causing a phase shift between the test and reference beams. Thesystem records the irradiance of the resulting interference pattern atmany different phase shifts and then converts the irradiance to phasewavefront data by integrating the irradiance data using a PSI algorithm.The phase data are processed to remove phase ambiguities betweenadjacent pixels. Average optical thickness measurements for the regionsof interest A1-A2 and B1-B2 were calculated by averaging the opticalthickness across all pixels within the region, followed by subtractingthe average thickness value from a similarly-sized region adjacent tothe cell containing no material. The external region served as a localreference for zero optical thickness. To determine the response to thecyclicly-applied force (f=0.05 Hz), the time varying data, sampled at 2second intervals, was bandpass filtered around a center frequency f=0.05Hz.

Cell Chamber.

The cell chamber body was constructed from machined non-magneticstainless steel. Resistive heating elements with internal thermistors,driven by a feedback controlled power supply, were used to regulate thechamber temperature to within 0.5 degrees of 37 C. The fluid sample wascontained within a 13 mm diameter, 0.7 mm thick sub chamber, having a 1mm thick optical window on top and a 0.2 mm thick silicon floor.

Microspheres.

Micron-sized elemental nickel microspheres were obtained from DukeScientific as a dry powder. An aqueous suspension of microspheres wasdiluted 4:1 with 0.2% poly-L-lysine aqueous solution (Sigma) to inhibitaggregation and improve adhesion to the cell body. This microreflectorsolution was shaken vigorously before application to suspend anysedimented particles and reduce aggregates. 200 μL of the suspension waspipetted onto the sample and the microspheres allowed to settle for 1minute.

Magnetic Force Control.

Magnetic force was applied to the microspheres using a cylindrical rareearth magnet 7 mm in diameter by 21 mm long, oriented axially along thevertical direction below the test chamber. The magnet was positionedwith a feedback controlled motorized micrometer, capable of <10 μmaccuracy. The magnitude of magnetic flux perpendicular to the verticalaxis, as a function of axial distance, was measured with a miniatureHall probe (2×2 mm) and a F. W. Bell 5180 gaussmeter accurate to 0.1 G.In the “off” position, the magnet was lowered to >4 cm below the sample,resulting in negligible field at the sample point. The magnet waspositioned coaxially with the optical axis to ensure a uniform magneticflux across the viewing area (˜300×300 μm with the 20× objective). Theforce applied to the nickel microspheres as a function of magnetposition was determined using microcantilever arrays tipped withelemental nickel or several uniformly magnetic microspheres (Compel 8 umcarboxylated microspheres, Bangs Labs). Each microcantilever is 500microns long by 100 microns wide and 0.9 microns thick, with a nominalspring constant of 0.01 N/m. These commercially available arrays wereproduced by the IBM Zurich Research Laboratories using a proprietary dryetch, silicon-on-insulator (SOI) process. Using the optical profiler,the deflection of the reference cantilever could be determined to betterthat 1 nm. The volume magnetic moment for pure nickel (55 emu/g) wasassumed for both the microspheres and the nickel film deposited on thecantilever tips. Pure nickel is completely magnetically polarized atfield strengths of 200 G and higher, while the lowest field strengthused in measurements was 500 G. Preceding measurements, the magnet wasraised to with 1.5 mm of the sample, corresponding to a ˜2 kG flux atthe sample point, to ensure that the microspheres' magnetic moments wereoriented axially.

Discussion of the Effect of Microsphere Size on Cell Dynamics

In general, micron-sized magnetic microspheres (˜5 microns) of are awell tested method used to apply mechanical stimulation to mammaliancells in culture (see, e.g. Trepat et al., Nature 2007, 447, 592-U7;Fernandez et al., Biophys. J. 2006, 90, 3796-3805; Trepat et al.,Journal of Applied Physiology 2005, 98, 1567-1574; Fisher et al., Rev.Sci. Instrum. 2005, 76; de Vries et al., Biophys. J. 2005, 88,2137-2144; Lenormand et al., J. R. Soc. Interface 2004, 1, 91-97; Hu etal., American Journal of Physiology-Cell Physiology 2004, 287,C1184-C1191; Deng et al., American Journal of Physiology-Cell Physiology2004, 287, C440-C448; Hu et al., American Journal of Physiology-CellPhysiology 2003, 285, C1082-C1090; Mijailovich et al., Journal ofApplied Physiology 2002, 93, 1429-1436; Fabry et al., Journal of AppliedPhysiology 2001, 91, 986-994; Bausch et al., Biophys J 2001, 80,2649-57; Fabry et al., Journal of Magnetism and Magnetic Materials 1999,194, 120-125; Bausch et al., Biophys. J. 1999, 76, 573-579; and Bauschet al., Biophys. J. 1998, 75, 2038-2049, the contents of which areincorporated by reference).

Other methods with larger physical footprints and/or larger forces havealso been used successfully. These include whole cell aspiration intomicropipettes (see, e.g. Hochmuth et al., J. Biomech. 2000, 33, 15-22)glass micro-needles and punch indentation (see, e.g. Desprat et al.,Biophys. J. 2005, 88, 2224-2233; Engler et al., Surf. Sci. 2004, 570,142-154; Koay et al., J. Biomech. Eng.-Trans. ASME 2003, 125, 334-341;Shin et al., J. Orthop. Res. 1999, 17, 880-890; and Thoumine et al., J.Cell Sci. 1997, 110, 2109-2116), substrate pulling (see, e.g. Hayakawaet al., Experimental Cell Research 2001, 268, 104-114; Wang et al.,Annals of Biomedical Engineering 2005, 33, 337-342; and Schaffer et al.,J. Orthop. Res. 1994, 12, 709-719), fluid shear (see, e.g. Li et al.,Journal of Biological Chemistry 1997, 272, 30455-30462; and Tseng etal., Circulation Research 1995, 77, 869-878. and AFM cantilevers (see,e.g., Mahaffy et al., Biophys. J. 2004, 86, 1777-1793; Radmacher M.Measuring the elastic properties of living cells by the atomic forcemicroscope. In: Jena B, Horber J K H, editors. atomic force microscopyin cell biology; 2002. p. 67-87; and Rotsch C et al., Biophys. J. 2000,78, 520-535). In fact standard (sharp) AFM tips generate the largestlocal strains among all these methods. Dimitriadas et al (see, e.g.Dimitriadis et al., Biophys. J. 2002, 82, 2798-2810) have studied thisissue and concluded that cell indentation studies with AFM should beconducted with blunted tips or micron-sized microspheres glued to thecantilever. Despite this issue, regular sharpened AFM tips have beenwidely and successfully used in mechanical studies of cell physiology.In the case of our study, we are interested in stimulating the corecytoskeleton of the cell, and this requires an indenter withmicron-sized dimensions, and forces of 100 pN to 1 nN.

In this case, we are intentionally perturbing the cell mechanically inorder to observe the response of the cell body in regions adjacent tothe site of applied force. We are using two 5 micron diameter nickelmicrospheres, but the actual mode of perturbation is not critical nor isit the focus of our study. Rather it is the response of the cellfollowing perturbation. Alternatively, we could have used an AFM probe,optical tweezers or a micropipette to stimulate the cell. Were itrequired, the nickel microsphere could be removed from the cell surfacemagnetically, after stimulation, thought we do not believe this isnecessary.

Regarding the force levels applied here in comparison to othertechniques: Optical tweezers typically operate in the 100 fN to 100 pNrange. Magnetic bead-based tweezers operate in the pN to nN range. AFMtypically operates in the 100 pN and larger force range. In our case, weare applying ˜200 pN of force on a 5 micron diameter bead. At smallindentations (˜500 nm) this amounts to less than 1 pN applied for persquare nanometer. For comparison, the typical force generated by asingle molecular motor is in the range of several pN, while thecontractile force generated through the cytoskeleton of a whole cell mayrange from nN to mN. Antibody-antigen binding forces are on the order ofnN.

This subject is summarized in an excellent review article by K. VanVliet, G. Bao and Suresh: The biomechanics toolbox: experimentalapproaches for living cells and biomolecules,” Acta Materialia 51 (2003)5881-5905.

Example 3: Grin Spherical Mirror: A General Optical Probe Useful withEmbodiments of the Invention

A wide variety of micromirrors can be used with embodiments of theinvention, including for example commercially available micron-sizedelemental nickel microspheres and the like. In addition, one can use agradient index (GRIN) retro reflector device with very wide viewingangle and high reflectivity. While not confined to uses withinterferometry, these micromirror embodiments can make an ideal probefor very accurate interferometric measurements. In addition, whiletypically a micromirror, the GRIN mirror can be of any size, down tonanometers in diameter.

Such GRIN mirrors can be manufactured to have a variety of propertieswhich allow them to be used in a variety of contexts. For example, theseoptical probes can be sensitive to magnetic, electric and gravitationalfields. Such probes can be used for example: (1) to sense seismic wavesor gravity waves; (2) as a metrology device for manufacturing and thelike; (3) in combination with other apparatuses such as amicrocantilever sensor; (4) as a component in optical communicationsdevices; (4) as an optical switch; (5) to modify the force generated byoptical tweezers; and (6) as an atomic force microscope-like probe.

Descriptions of a number of typical embodiments of this invention areprovided below.

Background and Details Nano Mirror.

Nano mirrors described herein can have a range of diameters and may besmaller than the spot size of the LED beam used for the readout(usually >5 microns). Typically they could be a micrometer or larger,although even smaller mirrors could be used. The nano mirror can befabricated using a variety of techniques including Silicon or SiNxmicromachining. Other methods include the deposition of gold or othermetals through shadow masks combined with lift off techniques where theback of the substrate is dissolved away. In a batch process, millions ofmirrors may be readily created on one substrate. Contact printing, dippen lithography etc, all provide possible routes for mirror fabrication.The mirrors can be spherical, circular, square or may have a complex3-dimensional form as determined by the experiment and manufacturingprocesses.

The mirrors themselves are readily functionalized using thiol- orsilane-based chemistries. Such embodiments of the invention allow themirrors to be able to be bio-functionalized so that, for example, theyselectively attach themselves to a particular cell or cell type.

FIGS. 14A-14E provide schematics of illustrative processes useful tomake micro/nano mirror embodiments of the invention.

Design.

Embodiments of these nano mirrors can have strong reflective propertiesfrom a wide range of angles to the incident beam. Both of theseattributes can be crucial for a robust, easy to use measurement system.A large reflected signal enables a wide range of detector lensgeometries to be used and greatly simplifies the procedures needed tocontrol unwanted environmental backscatter. A wide viewing angle iscritical because the nano mirror orientation, when bound to a cellmembrane, cannot be controlled precisely. Such nano mirror embodimentscan use a gradient index (GRIN) spherical lens design, with onehemisphere partially coated with gold to provide a reflective surface.This design is known as a Lunberg lens, and is used commonly for radarreflectors and antennas but not for a nano-scale optical reflectora.

A Luneberg lens is spherical, with a radially varying index ofrefraction (N), such that an entering signal will be refracted into anearly elliptical path to a point on the opposite surface of the sphere(FIG. 15A). A reflective material located at the opposition point causesa return of the signal, again refracted elliptically through the sphereand out again in the direction it came. The radial index of refractionis given by the Equation below, where N_(surface)=that of thesurrounding medium.

N(r)=[2−(r/r′)²]^(1/2)

A Luneberg lens performs much as a parabolic reflector, except thatwhereas a parabolic reflector is effective along its central axis (X),the Luneberg lens is an effective retro reflector over a wide range ofentry angels (such as P′X′ to P″X″ in FIG. 15A).

The Luneberg lens reflector has close to a 55-degree viewing angle fromcenter, compared to about ˜5 degrees for a flat plate and ˜25 degreesfor corner reflectors. Its reflectivity approaches that of a flatsurface, about three times more reflective than a square trihedralcorner reflector and more than 200 times that of a metal sphere with thesame radius. These properties assume that the reflector is above theRayleigh wavelength limit. Using incident light of wavelength 450 nm,the lower limit for the mirror's diameter is approximately 900nanometers. In this regime, the reflectivity increases as a function ofr². For diameters smaller than about 200 nm, the reflectance dropsrapidly, proportional to r⁶. In typical embodiments of the invention,the spheres are in the range of 2-5 microns in diameter, similar to thesize of marker beads used in other ‘magnetic tweezer’ cellular studies.

A 6-shell sphere, with refractive index decreasing with each layer fromcenter, approximates the ideal Luneberg lens (see, e.g., FIG. 15B).Sanford and Sakurai calculated and subsequently demonstratedexperimentally, that this design, when the diameter is roughly fivewavelengths or larger, produces a close to ideal retro reflector whencapped with a conducting layer (see, e.g. Sakurai, H., M. Ohki, et at.(2000) International Journal of Infrared and Millimeter Waves 21(70):16341652). In fact, this multi-shell design is common in Luneberg lensreflectors used at microwave frequencies. Translating these parametersto optical frequencies (450 nm), a 2.5-micron diameter sphere, in water,produces the dimensions given Table 1 below.

TABLE 1 Nano-structured Six-shell Luneberg lens Lambda (nm) 450 Diameterlimit (nm) 2,580 Particle Diameter (nm) 2,500 Medium index of refraction1.33 Layer 1 2 3 4 5 6 Radius (nm) 658 790 921 1,053 1,184 1,250 Shellthickness (nm) 658 133 131 131 131 66 Index of refraction 2.47 2.21 2.041.84 1.60 1.40

Layer-by-Layer Fabrication.

Embodiments of the invention can be made by a variety of processes knownin the art, such as the layer-by-layer multishell nanoparticle methodsused to fabricate Luneberg lens reflectors (see, e.g. Caruso, et al.(1998) Science 282(5391): 1111-1114). Such layer-by-layer techniquestypically involve the consecutive adsorption of material on a sphericalcore to produce a nano-structured particle. Such methods have been usedwith a wide variety of materials and combinations, including organic andinorganic polymers, semiconductors, metals, and biomolecules ofdifferent types. The techniques have progressed to such a degree thatthe thickness, composition and uniformity of each layer is highlycontrollable. Studies to date have largely focused on the use ofmulti-shell particles in optoelectronics and in drug delivery. Particlescan be selected by simple filtering at each coating stage to assureuniformity.

In a typical nanomirror embodiment, each layer can be composed of amixed polymer material with the desired index of refraction. The nanomirror will be working in a liquid (cell culture medium) with an indexof refraction similar to water, which is 1.3333, making surface indexmatching a non-issue. Ideally, a center index should be 2.5. One optionfor obtaining this is to nucleate the sphere with a very small (o 50nm), high index polysilicon particle, which at 450 nm has an index ofrefraction around five and an extinction coefficient close to zero.Fortunately, very high index polymers, with N up to about 2.2, are nowavailable commercially (e.g. BREWER SCIENCE'S Specialty MaterialsDivision). These transparent polymer-coating materials usually consistof multiple polymers species, with one component conjugated to a highrefractive index metal oxide. The refractive index of the coating isthen variable, depending on the ratio of the different components.

A next typical fabrication step is to vapor deposit reflective gold capon the nanostructured polymer sphere. Artisans can assemble a monolayerof the spheres, using standard techniques, on silicon or some othersurface. In this configuration, using latex micro beads, we have shownthat the gold coating is confined to the hemisphere opposite the solidsurface. If further control over the gold deposition is required, we canblock the lower part of the sphere with a soluble, inert filler layer inthe interstices between the spheres. In the final step, we will vapordeposit a layer of ferromagnetic material on top of the gold cap,followed by another thin layer of gold. The final gold layer will allow,via thiol linker chemistry, functionalization with fibronectin or someother cell adhesive substance.

A variety of methods and materials known in the art can be adapted tomake and/or use embodiments of the invention, for example thosedisclosed in Abdelsalam, et al. (2004): Advanced Materials 16(1): 90−+;Alenohat et al. (2000): Biochem Biophys Res Commun 277(1): 93-9;Allersma et al. (1998): Biophysical Journal 74(2): 1074-1085; Bartlett,et al. (2004): Faraday Discussions 125: 11 7-1 32; Bausch et al. (2001):Biophys J 80(6): 2649-57; Caruso et al. (1998): Science 282(5391):1111-1114; Caruso et al., (1999): Langmuir 15(23): 8276-8281; Caruso etal. (1999): Chemistry of Materials 11(11): 3394-3399; Caruso et al.(2001): Chemistry of Materials 13(2): 400-409; Coyle et al. (2003):Applied Physics Letters 83(4): 767-769; Gallet, F. (2004): Annales DeBiologie Clinique 62(1): 85-86; Gittins et al., (2001): Journal ofPhysical Chemistry B 105(29): 6846-6852; Goldschmidt et al. (2001): CircRes 88(7): 674-80; Guck et al. (2001): Biophys J 81(2): 767-84; Hannayet al., (1993): Journal of Modern Optics 40(8): 1437-1442; Hardaker etal., (1994): Journal of Electromagnetic Waves and Applications 8(3):391-405; Kato et al. (2002): Macromolecules 35(26): 9780-9787; Koike etal. (1986): Applied Optics 25(19): 3356-3363; Liang et al. (2003):Chemistry of Materials 15(16): 3176-3183; Pommerenke et al. (1996): EurJ Cell Biol 70(2): 157-64; Schuetz et al., (2003): Advanced FunctionalMaterials 13(12): 929-937; Seward et al., (1999): Optical Engineering38(1): 164-169; and Wang et al. (2002): Nano Letters 2(8): 857-861, thecontents of each of which is incorporated by reference.

Magnetically Driven Optical Bead Probe for In Situ Diagnosis of Cancer

One embodiment of the invention as shown in FIG. 16 combines the use ofan external magnetic field to exert a force on a small optical bead withmagnetic properties which uses specially graded index glass and areflector to detect the mechanical response of the bead. This providesan in vivo measuring of the local mechanical properties and motility ofcellular motion with a spatial resolution determined by the diameter ofthe bead. The bead can be tethered to a thin connecting wire which isflexible and connects to a rod on the end of a hypodermic needle. Themagnetic drive and the needle assembly fit easily into even the smallestendoscope devices.

The rod connecting the optical sphere is fabricated from a singleoptical fiber which transmits and detects light in an interferometermode of operation. In this embodiment of the invention, the local motionof the cell with respect to the fiber end is measured. Specifically, onthe relative motion of the bead with the end of the fiber is detectedwhich makes the device capable of removing background signals from organmotion possible. The length of the attachment cable between bead andfiber them determines also the spatial resolution of the device.

In the operational mode a surgeon or doctor probes local tissue with theneedle directed by endoscope of other means such as ultrasound or X-rayimaging. The device constantly measures the local mechanical propertiesof the tissue. Cancerous cells are much softer and they are detected bythe device using a computer technique programmed through neural learninga logarithms for different tissues (e.g. breast cancer etc.). Once thecancerous tissue is mapped and located, the position of the bead can bedetected by having a fluorescent component of by external imagingmethods or by position interpolation routines. This mapping can becombined by a variety of in vivo treatments which could involve removalof the probing sphere and fiber assembly and injection of chemotherapyagents or laser ablation for example.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-10. (canceled) 11: A method for observing changes in mammalian cellmass using an interference microscope, the method comprising: (a)placing a mammalian cell in an observation chamber of the interferencemicroscope, wherein the observation chamber is adapted to form aqueousenvironments; (b) using phase shifting interferometry to observe phaseimages of the mammalian cell in a first environment; (c) altering thefirst environment to form a second environment; (d) using phase shiftinginterferometry to observe phase images of the mammalian cell in thesecond environment; (e) comparing the phase images observed in (b) withthe phase images observed in (d) so as to observe changes in the cellmass of the mammalian cell, so that changes in mammalian cell mass areobserved. 12: A system for obtaining an image of a cell comprising: (a)microscope capable of measuring a feature of interest in a sample; (b) adetector operatively coupled to the microscope; (c) a sample assemblycomprising an observation chamber adapted to contain the cell; (d) areference assembly comprising: a first optical window; a first housingelement adapted to hold the first optical window; a second opticalwindow; a second housing element adapted to hold the second opticalwindow; and a plurality of spacer elements disposable between the firstoptical window and the second optical window and adapted to separate thefirst and second optical windows to a defined distance. 13: The systemof claim 12, wherein the system further comprises at least one perfusionconduit adapted to introduce a composition into the observation chamber.14: The system of claim 12, wherein the microscope is an interferencemicroscope capable of observing interference fringes. 15: The system ofclaim 12, wherein the sample assembly further comprises: a viewingwindow; a first housing element adapted to hold the viewing window; andwherein the thickness of the viewing window is equivalent to thecombined thickness of the first and second optical windows in thereference assembly. 16: The system of claim 12, wherein the systemcomprises: a memory storage element adapted to store one or more imagesof the cell; and a processor element adapted to process one or moreimages of the cell. 17: The system of claim 12, wherein the systemcomprises: a sample assembly comprising an observation chamber adaptedto contain the cell; and a reference assembly comprising a referencechamber adapted to contain a fluid. 18: The system of claim 12, whereinthe observation chamber comprises a human cell obtained from a biopsy.19: The system of claim 12, wherein the system further comprises atleast one composition that alters a cell mass property of a cell. 20:The system of claim 12, further comprising a magnet disposed below theobservation chamber and oriented coaxially with an optical axis. 21: Themethod of claim 11, wherein the method comprises observing changes in atleast one of: an optical thickness of the mammalian cell; a cellulardensity of the mammalian cell; a volume of the mammalian cell; an indexof refraction for the mammalian cell; and a viscoelastic property of themammalian cell. 22: The method of claim 11, wherein the method observeschanges in mammalian cell mass that occur following combining themammalian cell with a composition comprising a biologically activeagent. 23: The method of claim 22, wherein the method is used to observea response to stimuli in a plurality of mammalian cells. 24: The methodof claim 22, wherein the method is used to observe changes in mammaliancell mass under changing environmental conditions over time. 25: Themethod of claim 11, wherein the wherein the microscope is aninterference microscope capable of observing interference fringes. 26:The method of claim 11, wherein the method is performed using a systemcomprising: (a) a detector operatively coupled to the microscope; (b) asample assembly comprising the observation chamber; (c) a referenceassembly comprising a reference chamber adapted to contain a fluid; (d)a memory storage element adapted to store one or more images of thecell; and (e) a processor element adapted to process one or more imagesof the cell. 27: The method of claim 26, wherein the microscope includesa reference assembly comprising: a first optical window; a first housingelement adapted to hold the first optical window; a second opticalwindow; a second housing element adapted to hold the second opticalwindow; and a plurality of spacer elements disposable between the firstoptical window and the second optical window and adapted to separate thefirst and second optical windows to a defined distance. 28: The methodof claim 11, wherein the method comprises obtaining information on acell specific profile of a live cell in an aqueous medium and storingthis information in the memory storage element. 29: The method of claim11, further comprising removing the cell from the observation chamberand manipulating the cell for further analysis.